U.S. patent application number 09/974760 was filed with the patent office on 2003-07-31 for iove variant regulator molecules.
Invention is credited to Milne, G. Todd, Roberts, Shannon, Sherman, Amir, Trueheart, Joshua.
Application Number | 20030143705 09/974760 |
Document ID | / |
Family ID | 25522409 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143705 |
Kind Code |
A1 |
Roberts, Shannon ; et
al. |
July 31, 2003 |
IovE variant regulator molecules
Abstract
The invention provides variant regulator proteins of secondary
metabolite production and nucleic acids encoding said variant
regulator proteins. In particular, the invention provides variant
regulator molecules of the lovE protein.
Inventors: |
Roberts, Shannon;
(Cambridge, MA) ; Sherman, Amir; (Jerusalem,
IL) ; Trueheart, Joshua; (Concord, MA) ;
Milne, G. Todd; (Brookline, MA) |
Correspondence
Address: |
Wayne A. Keown, Ph.D.
Suite 2900
500 W. Cummings Park
Woburn
MA
01801-6544
US
|
Family ID: |
25522409 |
Appl. No.: |
09/974760 |
Filed: |
October 9, 2001 |
Current U.S.
Class: |
435/183 |
Current CPC
Class: |
C07K 14/38 20130101 |
Class at
Publication: |
435/183 |
International
Class: |
C12N 009/00 |
Claims
What is claimed is:
1. A protein comprising an amino acid sequence that codes for a
variant protein of the lovE protein having at least one mutation
selected from the group consisting of: (a) a Group 6 amino acid
residue mutated to a Group 2 amino acid residue at position 31; (b)
a Group 3 amino acid residue mutated to a Group 5 amino acid
residue at position 41; (c) a Group 4 amino acid residue mutated to
a Group 2 amino acid residue at position 52; (d) a Group 4 amino
acid residue mutated to a Group 3 amino acid residue at position
52; (e) a Group 4 amino acid residue mutated to a Group 5 amino
acid residue at position 73; (f) a Group 1 amino acid residue
mutated to a Group 4 amino acid residue at position 101; (g) a
Group 1 amino acid residue mutated to a Group 3 amino acid residue
at position 101; (h) a valine amino acid residue mutated to another
Group 2 amino acid residue at position 111; (i) a Group 4 amino
acid residue mutated to a Group 2 amino acid residue at position
133; (j) a Group 3 amino acid residue mutated to a Group 2 amino
acid residue at position 141; (k) a Group 3 amino acid residue
mutated to a Group 5 amino acid residue at position 141; (l) a
Group 4 amino acid residue mutated to Group 6 amino acid residue at
position 153; (m) a Group 4 amino acid residue mutated to a Group 5
amino acid residue at position 153; (n) a Group 4 amino acid
residue mutated to a Group 1 amino acid residue at position 281;
(o) a Group 3 amino acid residue mutated to a Group 2 amino acid
residue at position 367; (p) a Group 3 amino acid residue mutated
to a Group 6 amino acid residue at position 367; (q) a Group 1
amino acid residue mutated to Group 4 amino acid residue at
position 389; and (r) a Group 1 amino acid residue mutated to a
Group 2 amino acid residue at position 389.
2. The protein of claim 1, wherein the variant protein has a Group
6 amino acid residue mutated to a Group 2 amino acid residue at
position 31.
3. The protein of claim 2 having the mutation F31L.
4. The protein of claim 1, wherein the variant protein has a Group
3 amino acid residue mutated to a Group 5 amino acid residue at
position 41.
5. The protein of claim 4 having the mutation Q41K or Q41R.
6. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to a Group 2 amino acid residue at
position 52.
7. The protein of claim 6 having the mutation T52I.
8. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to a Group 3 amino acid residue at
position 52.
9. The protein of claim 8 having the mutation T52N.
10. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to a Group 5 amino acid residue at
position 73.
11. The protein of claim 10 having the mutation C73R.
12. The protein of claim 1, wherein the variant protein has a Group
1 amino acid residue mutated to a Group 4 amino acid residue at
position 101.
13. The protein of claim 12 having the mutation P101S.
14. The protein of claim 1, wherein the variant protein has Group 1
amino acid residue mutated to a Group 3 amino acid residue at
position 101.
15. The protein of claim 14 having the mutation P101Q.
16. The protein of claim 1, wherein the variant protein has a
valine amino acid residue mutated to another Group 2 amino acid
residue at position 111.
17. The protein of claim 16 having the mutation V111I.
18. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to a Group 2 amino acid residue at
position 133.
19. The protein of claim 18 having the mutation S133L.
20. The protein of claim 1, wherein the variant protein has Group 3
amino acid residue mutated to a Group 2 amino acid residue at
position 141.
21. The protein of claim 20 having the mutation E141V.
22. The protein of claim 1, wherein the variant protein has a Group
3 amino acid residue mutated to a Group 5 amino acid residue at
position 141.
23. The protein of claim 22 having the mutation E141K.
24. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to Group 6 amino acid residue at
position 153.
25. The protein of claim 24 having the mutation C153Y.
26. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to a Group 5 amino acid residue at
position 153.
27. The protein of claim 26 having the mutation C153R.
28. The protein of claim 1, wherein the variant protein has a Group
4 amino acid residue mutated to a Group 1 amino acid residue at
position 281.
29. The protein of claim 28 having the mutation T281A.
30. The protein of claim 1, wherein the variant protein has Group 3
amino acid residue mutated to a Group 2 amino acid residue at
position 367.
31. The protein of claim 30 having the mutation N367I.
32. The protein of claim 1, wherein the variant protein has a Group
3 amino acid residue mutated to a Group 6 amino acid residue at
position 367.
33. The protein of claim 32 having the mutation N367Y.
34. The protein of claim 1, wherein the variant protein has a Group
1 amino acid residue mutated to Group 4 amino acid residue at
position 389.
35. The protein of claim 34 having the mutation P389S.
36. The protein of claim 1, wherein the variant protein has a Group
1 amino acid residue mutated to a Group 2 amino acid residue at
position 389.
37. The protein of claim 36 having the mutation P389L.
38. The protein of claim 1 selected from the group consisting of
SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ
ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57,
SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID
NO:63, SEQ ID NO:64, and SEQ ID NO:65.
39. A nucleic acid comprising a polynucleotide sequence encoding an
amino acid sequence of a variant protein of the lovE protein having
at least one mutation selected from the group consisting of: (a) a
Group 6 amino acid residue mutated to a Group 2 amino acid residue
at position 31; (b) a Group 3 amino acid residue mutated to a Group
5 amino acid residue at position 41; (c) a Group 4 amino acid
residue mutated to a Group 2 amino acid residue at position 52; (d)
a Group 4 amino acid residue mutated to a Group 3 amino acid
residue at position 52; (e) a Group 4 amino acid residue mutated to
a Group 5 amino acid residue at position 73; (f) a Group 1 amino
acid residue mutated to a Group 4 amino acid residue at position
101; (g) a Group 1 amino acid residue mutated to a Group 3 amino
acid residue at position 101; (h) a valine amino acid residue
mutated to another Group 2 amino acid residue at position 111; (i)
a Group 4 amino acid residue mutated to a Group 2 amino acid
residue at position 133; (j) an Group 3 amino acid residue mutated
to a Group 2 amino acid residue at position 141; (k) an Group 3
amino acid residue mutated to a Group 5 amino acid residue at
position 141; (l) a Group 4 amino acid residue mutated to Group 6
amino acid residue at position 153; (m) a Group 4 amino acid
residue mutated to a Group 5 amino acid residue at position 153;
(n) a Group 4 amino acid residue mutated to a Group 1 amino acid
residue at position 281; (o) a Group 3 amino acid residue mutated
to a Group 2 amino acid residue at position 367; (p) a Group 3
amino acid residue mutated to a Group 6 amino acid residue at
position 367; (q) a Group 1 amino acid residue mutated to Group 4
amino acid residue at position 389; and (r) a Group 1 amino acid
residue mutated to a Group 2 amino acid residue at position
389.
40. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 6
amino acid residue mutated to a Group 2 amino acid residue at
position 31.
41. The nucleic acid of claim 40 having the mutation F31L.
42. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 3
amino acid residue mutated to a Group 5 amino acid residue at
position 41.
43. The nucleic acid of claim 42 having the mutation Q41K or
Q41R.
44. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to a Group 2 amino acid residue at
position 52.
45. The nucleic acid of claim 44 having the mutation T52I.
46. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to a Group 3 amino acid residue at
position 52.
47. The nucleic acid of claim 46 having the mutation T52N.
48. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to a Group 5 amino acid residue at
position 73.
49. The nucleic acid of claim 48 having the mutation C73R.
50. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 1
amino acid residue mutated to a Group 4 amino acid residue at
position 101.
51. The nucleic acid of claim 50 having the mutation P101S.
52. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having Group 1 amino
acid residue mutated to a Group 3 amino acid residue at position
101.
53. The nucleic acid of claim 52 having the mutation P101Q.
54. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a valine amino
acid residue mutated to another Group 2 amino acid residue at
position 111.
55. The nucleic acid of claim 54 having the mutation V111I.
56. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to a Group 2 amino acid residue at
position 133.
57. The nucleic acid of claim 56 having the mutation S133L.
58. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having Group 3 amino
acid residue mutated to a Group 2 amino acid residue at position
141.
59. The nucleic acid of claim 58 having the mutation E141V.
60. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 3
amino acid residue mutated to a Group 5 amino acid residue at
position 141.
61. The nucleic acid of claim 60 having the mutation E141K.
62. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to Group 6 amino acid residue at
position 153.
63. The nucleic acid of claim 62 having the mutation C153Y.
64. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to a Group 5 amino acid residue at
position 153.
65. The nucleic acid of claim 64 having the mutation C153R.
66. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 4
amino acid residue mutated to a Group 1 amino acid residue at
position 281.
67. The nucleic acid of claim 66 having the mutation T281A.
68. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 3
amino acid residue mutated to a Group 2 amino acid residue at
position 367.
69. The nucleic acid of claim 68 having the mutation N367I.
70. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 3
amino acid residue mutated to a Group 6 amino acid residue at
position 367.
71. The nucleic acid of claim 70 having the mutation N367Y.
72. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 1
amino acid residue mutated to Group 4 amino acid residue at
position 389.
73. The nucleic acid of claim 72 having the mutation P389S.
74. The nucleic acid of claim 39, wherein the polynucleotide
encodes a variant protein of the lovE protein having a Group 1
amino acid residue mutated to a Group 2 amino acid residue at
position 389.
75. The nucleic acid of claim 74 having the mutation P389L.
76. The nucleic acid of claim 39 selected from the group consisting
of SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ
ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81,
SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID
NO:87, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to the fields of microbiology and
molecular biology. In particular, the invention relates to the
field of mycology and the production of secondary metabolites from
fungi.
[0003] 2. Summary of the Related Art
[0004] Secondary metabolites are a major source of commercially
useful products such as food additives, vitamins, and medicines for
the treatment of a wide variety of infections and diseases. By way
of example, in 1997 the statin drugs lovastatin, simvastatin, and
pravastatin, fungal secondary metabolites used in the treatment of
hypercholesteremia, together had US sales of US$7.53 billion
(Sutherland et al., Current Opinion In Drug Discovery &
Development 4:229-236 (2001)). The cost and availability of these
plant, bacterial and fungal metabolites are frequently determined
by limitations imposed on production and purification of these
compounds from culture. This problem is frequently exacerbated by
the fact that these products are generally produced during the
stationary phase of bacterial and fungal growth.
[0005] A wide variety of methods have been utilized to increase the
amount of secondary metabolite produced in culture. Studies have
demonstrated the importance of carefully designing the medium in
which a fungus is grown to maximize the amount of a secondary
metabolite produced (see, e.g., Hajjaj H, et al., Appl. Environ.
Microbiol. 67:2596-602 (2001); Lesova, K., et al., J. Basic
Microbiol. 40:369-75 (2000)). In addition, the method of culture or
fermentation also impacts directly on the amount of secondary
metabolite produced. For example, see Robinson, T., et al. (Appl.
Microbiol. Biotechnol. 55:284-289 (2001)), which demonstrates the
advantages of solid state (substrate) fermentation.
[0006] In addition to the manipulation of culture and media
conditions, genetic approaches have been taken to increase
secondary metabolite production. For example, the production of
penicillin is limited by the activity of two enzymes, encoded by
the ipnA and acvA genes, both of which are regulated by the pacC
protein, a zinc-finger transcription factor. Naturally occurring
mutant alleles of the pacC locus are known to possess more
transcription-activating activity than the cognate, wild-type
allele (see, e.g., Tilburn et al. EMBO J. 14(4):779-790 (1995)).
Thus, one genetic approach to increasing secondary metabolite
production is to identify and isolate naturally occurring mutant
alleles, the expression of which leads to increased secondary
metabolite production.
[0007] Although many regulators of secondary metabolite production
in many organisms are known, not all of the organisms that produce
secondary metabolites are amenable to genetic or molecular genetic
manipulation. Thus, these systems are not generally useful as a
source for the isolation of naturally occurring mutant alleles and
are even less useful for the deliberate manipulation of secondary
metabolite regulator protein structure with the aim of creating
improved regulators of secondary metabolite production.
[0008] It would be advantageous to have improved regulators of the
biosynthetic enzymes responsible for secondary metabolite
production. For example, recent studies suggest increasing usage of
statin drugs, e.g., see Waters D. D., Am. J. Cardiol. 88:10F-5F
(2001)). Thus, demand for statin drugs is likely to increase
substantially. In order to meet the demand for these and other
secondary metabolites, new and improved methods for the production
of secondary metabolites must be identified.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides improved secondary metabolite
regulator proteins that enable increased production of secondary
metabolites. The invention also provides methods to make these
improved regulator proteins.
[0010] In a first aspect, the invention provides a variant
regulator protein of secondary metabolite production with increased
activity than that of the cognate, wild-type protein. In certain
embodiments of this aspect of the invention, the regulator protein
is a fungal regulator protein.
[0011] In an embodiment of the first aspect, the invention provides
an improved regulator protein comprising an amino acid sequence
coding for a variant lovE protein having at least one specific
mutation that gives rise to greater transcription-activating
properties of the regulator protein and/or induction of secondary
metabolite synthesis.
[0012] By way of non-limiting example, certain preferred regulator
proteins of this aspect of the invention include at least one of
the following mutations: (1) a Group 6 amino acid residue mutated
to a Group 2 amino acid residue at position 31, in one embodiment
the mutation represented by F31L; (2) a Group 3 amino acid residue
mutated to a Group 5 amino acid residue at position 41, in one
embodiment the mutation represented by Q41K or Q41R; (3) a Group 4
amino acid residue mutated to a Group 2 amino acid residue at
position 52, in one embodiment the mutation represented by T52I;
(4) a Group 4 amino acid residue mutated to a Group 3 amino acid
residue at position 52, in one embodiment the mutation represented
by T52N; (5) a Group 4 amino acid residue mutated to a Group 5
amino acid residue at position 73, in one embodiment the mutation
represented by C73R; (6) a Group 1 amino acid residue mutated to a
Group 4 amino acid residue at position 101, in one embodiment the
mutation represented by P101S; (7) a Group 1 amino acid residue
mutated to a Group 3 amino acid residue at position 101, in one
embodiment the mutation represented by P101Q; (8) a valine amino
acid residue mutated to another Group 2 amino acid residue at
position 111, in one embodiment the mutation represented by V111I;
(9) a Group 4 amino acid residue mutated to a Group 2 amino acid
residue at position 133, in one embodiment the mutation represented
by S133L; (10) a Group 3 amino acid residue mutated to a Group 2
amino acid residue at position 141, in one embodiment the mutation
represented by E141V; (11) a Group 3 amino acid residue mutated to
a Group 5 amino acid residue at position 141, in one embodiment the
mutation represented by E141K; (12) a Group 4 amino acid residue
mutated to Group 6 amino acid residue at position 153, in one
embodiment the mutation represented by C153Y; (13) a Group 4 amino
acid residue mutated to a Group 5 amino acid residue at position
153, in one embodiment the mutation represented by C153R; (14) a
Group 4 amino acid residue mutated to a Group 1 amino acid residue
at position 281, in one embodiment the mutation represented by
T281A; (15) a Group 3 amino acid residue mutated to a Group 2 amino
acid residue at position 367, in one embodiment the mutation
represented by N367I; (16) a Group 3 amino acid residue mutated to
a Group 6 amino acid residue at position 367, in one embodiment the
mutation represented by N367Y; (17) a Group 1 amino acid residue
mutated to Group 4 amino acid residue at position 389, in one
embodiment the mutation represented by P389S; and (18) a Group 1
amino acid residue mutated to a Group 2 amino acid residue at
position 389, in one embodiment the mutation represented by
P389L.
[0013] In some embodiments of the first aspect, the invention
provides regulator proteins with at least two, or at least three,
or at least four, or at least five, or at least six, or at least
seven, or at least eight, or at least nine, or at least ten, or at
least eleven, or at least twelve, or at least thirteen, or at least
fourteen, or at least fifteen, or at least sixteen, or at least
seventeen, or at least eighteen of the above described specific
mutations.
[0014] In other embodiments of the first aspect, the invention
provides an isolated lovE variant regulator protein selected from
the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,
SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID
NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ
ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57,
SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65.
[0015] In a second aspect, the invention provides a nucleic acid
molecule encoding a lovE regulator of the first aspect of the
invention. By way of non-limiting example, the invention provides a
nucleic acid molecule encoding the lovE variant regulator protein
selected from the group consisting of SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID
NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ
ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83,
SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, and SEQ ID NO:90.
[0016] In a third aspect, the invention provides a method of
increasing the activity of a protein that regulates secondary
metabolite production comprising: (a) selecting a nucleic acid
comprising a polynucleotide encoding a protein regulator of
secondary metabolite production; (b) mutating the nucleic acid to
create a plurality of nucleic acid molecules encoding variant
regulator proteins of secondary metabolite production; and (c)
selecting a variant regulator protein with more activity than the
cognate, wild-type protein.
[0017] In various embodiments of the third aspect, the secondary
metabolite is a fungal secondary metabolite. In certain embodiments
of the third aspect, the protein regulator of secondary metabolite
production is a transcription factor. In certain embodiments of the
third aspect, the protein regulator of secondary metabolite
production is a transmembrane transporter, protein that mediates
secretion, kinase, G-protein, cell surface receptor, GTPase
activating protein, guanine nucleotide exchange factor,
phosphatase, protease, phosphodiesterase, bacterial protein toxin,
importin, RNA-binding protein, SCF complex component, adherin, or
protein encoded within a biosynthetic cluster. In certain other
embodiments of the third aspect, the variant regulator protein is
selected to have more activity in a heterologous cell and/or more
activity in a homologous cell than the cognate, wild-type regulator
protein. In certain embodiments, the variant regulator protein is
selected to have more activity in a heterologous cell and/or more
activity in a homologous cell than the cognate, wild-type protein
and to cause more secondary metabolite to be produced in a
homologous cell and/or a heterologous cell when compared to the
cognate, wild-type regulator protein. In a particularly preferred
embodiment, the variant regulator protein is a lovE variant
regulator protein.
[0018] In a fourth aspect, the invention provides a method of
increasing production of a secondary metabolite comprising: (a)
selecting a nucleic acid comprising a polynucleotide encoding a
protein regulator of secondary metabolite production; (b) mutating
the nucleic acid to create a plurality of nucleic acid molecules
encoding variant regulator proteins of secondary metabolite
production; (c) selecting a variant regulator protein with more
activity than the cognate, wild-type protein; and (d) expressing
the selected variant regulator protein in a cell, thereby
increasing production of the secondary metabolite in the cell.
[0019] In various embodiments of the fourth aspect, the secondary
metabolite is a fungal secondary metabolite. In certain embodiments
of the third aspect, the protein regulator of secondary metabolite
production is a transcription factor. In certain embodiments of the
fourth aspect, the protein regulator of secondary metabolite
production is a transmembrane transporter, a protein that mediates
secretion, a kinase, a G-protein, a cell surface receptor, a GTPase
activating protein, a guanine nucleotide exchange factor, a
phosphatase, a protease, a phosphodiesterase, a bacterial protein
toxin, an importin, an RNA-binding protein, an SCF complex
component, an adherin, or a protein encoded within a biosynthetic
cluster. In certain other embodiments of the fourth aspect, the
variant regulator protein is selected to have more activity in a
heterologous cell and/or more activity in a homologous cell. In
certain embodiments, the variant regulator protein is selected to
have more activity in a heterologous cell and/or more activity in a
homologous cell and to cause more secondary metabolite to be
produced in a homologous cell and/or a heterologous cell when
compared to the cognate, wild-type regulator protein. In a
particularly preferred embodiment, the variant regulator protein is
a lovE variant regulator protein.
[0020] In a fifth aspect, the invention provides an isolated
variant regulator protein of secondary metabolite production having
increased activity compared to a cognate, wild-type protein, the
variant regulator protein made by the process comprising: (a)
selecting a nucleic acid comprising a polynucleotide encoding a
protein regulator of secondary metabolite production; (b) mutating
the nucleic acid to create a plurality of nucleic acid molecules
encoding variant regulator proteins of secondary metabolite
production; (c) selecting a variant regulator protein with more
activity than the cognate, wild-type protein; and (d) recovering
the selected variant regulator protein.
[0021] In certain embodiments of the fifth aspect, the secondary
metabolite is a fungal secondary metabolite. In certain embodiments
of the fifth aspect, the protein regulator of secondary metabolite
production is a transcription factor. In certain embodiments of the
fifth aspect, the protein regulator of secondary metabolite
production is a transmembrane transporter, a protein that mediates
secretion, a kinase, a G-protein, a cell surface receptor, a GTPase
activating protein, a guanine nucleotide exchange factor, a
phosphatase, a protease, a phosphodiesterase, a bacterial protein
toxin, an importin, an RNA-binding protein, an SCF complex
component, an adherin, or a protein encoded within a biosynthetic
cluster. .In certain embodiments of the fifth aspect, the variant
regulator protein has more activity in a heterologous and/or a
homologous cell than the cognate, wild-type protein. In certain
embodiments of the fourth aspect, the variant regulator protein
increases production of a secondary metabolite in a heterologous
cell and/or a homologous cell when compared to the cognate,
wild-type protein. In a particularly preferred embodiment, the
variant regulator protein is a lovE variant regulator protein.
[0022] In a sixth aspect, the invention provides a fungus having
improved lovastatin production made by the process of transforming
a fungal cell with a nucleic acid molecule encoding a lovE variant
protein of the first aspect of the invention. In an embodiment
thereof, the nucleic acid molecule is selected from a nucleic acid
molecule of the second aspect of the invention.
[0023] In a seventh aspect, the invention provides an improved
process for making lovastatin comprising transforming a fungal cell
with a nucleic acid molecule encoding a variant of the lovE protein
of the first aspect of the invention. In an embodiment thereof, the
fungal cell is transformed with a nucleic acid molecule of the
second aspect of the invention.
[0024] In a eighth aspect, the invention provides a nucleic acid
molecule encoding a lovE protein defined by SEQ ID NO:91. In an
embodiment thereof, the invention provides an isolated lovE nucleic
acid molecule defined by SEQ ID NO:92.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a photographic representation of cells growing on
media with and without G418 selection demonstrating lovFp-HIS3p-Neo
activation in S. cerevisiae. Controls include MB968 (vector only),
MB2478 (lowly expressed wild-type lovE), and MB1644 (highly
expressed wild-type lovE). All lovE variants are expressed in an
MB968 vector backbone similar to MB2478.
[0026] FIG. 2A is a graphic representation of lovFp-CYC1p-lacZ
expression in S. cerevisiae strains expressing lovE variant
proteins from the clones lovE 1-10.
[0027] FIG. 2B is a graphic representation of lovFp-CYClp-lacZ
expression in S. cerevisiae strains expressing lovE variant
proteins from the clones lovE 1-10 from a separate transformation
than that of FIG. 2A.
[0028] FIG. 3 is a graphic presentation of lovFp-CYC1p-lacZ
expression in S. cerevisiae strains expressing lovE variant
proteins from clones lovE 16-41.
[0029] FIG. 4 is a graphic presentation of lovFp-lacZ expression in
S. cerevisiae strains expressing lovE variant proteins from clones
lovE 1-10.
[0030] FIG. 5 is a graphic presentation of lovFp-lacZ expression in
S. cerevisiae strains expressing lovE variant proteins from clones
lovE 16, 20, 21, 30-34, and 36-41.
[0031] FIG. 6 is a graphic presentation of lovastatin culture
concentration, as measured by enzyme inhibition assay, from broths
of A. terreus cultures expressing love variant proteins 1-10
in.
[0032] FIG. 7A is a graphic depiction of lovastatin culture
concentration, as measured by HPLC analysis, from broths of A.
terreus cultures expressing lovE variant proteins 1-10 in
MF117.
[0033] FIG. 7B is a graphic depiction of lovastatin culture
concentration, as measured by HPLC analysis, from broths of A.
terreus cultures expressing lovE variant proteins 2, 6, 30, 32, 36,
37, 39, and 41 in MF117.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0034] The patents and publications cited herein reflect the level
of knowledge in the art and are hereby incorporated by reference in
their entirety. Any conflict between any teaching of such
references and this specification shall be resolved in favor of the
latter.
[0035] The invention utilizes techniques and methods common to the
fields of molecular biology, genetics and microbiology. Useful
laboratory references for these types of methodologies are readily
available to those skilled in the art. See, for example, Molecular
Cloning, A Laboratory Manual, 3.sup.rd edition, edited by Sambrook,
J., MacCallum, P., and Russell, D. W. (2001), Cold Spring Harbor
Laboratory Press (ISBN: 0-879-69576-5); Current Protocols In
Molecular Biology, edited by Ausubel, F. M., Brent, R., Kingston,
R. E., Moore, D. D., Seidman, J. G., Struhl, K. (1993), John Wiley
and Sons, Inc. (ISBN: 0-471-30661-4); PCR Applications: Protocols
for Functional Genomics, edited by Innis, M. A., Gelfand, D. H.,
Sninsky, J. J. (1999), Cold Spring Harbor Press (ISBN:
0-123-72186-5); and Methods In Yeast Genetics, 2000 Edition: A Cold
Spring Habor Laboratory Course Manual, by Burke, D., Dawson, D. and
Stearns, T., Cold Spring Harbor Press (ISBN: 0-879-69588-9).
[0036] In certain embodiments of the aspects of the invention, the
invention relates to the biosynthesis and improved production of
secondary metabolites. The invention provides variant regulator
proteins useful for the production of secondary metabolites,
nucleic acid molecules encoding variant regulator proteins, and
methods for their production.
[0037] In a first aspect, the invention provides a variant
regulator protein of secondary metabolite production with increased
activity relative to a cognate, wild-type regulator protein.
Particularly preferred are variant regulator proteins of fungal
secondary metabolites.
[0038] As used herein, the terms "fungal" and "fungus" refer
generally to eukaryotic, heterotrophic organisms with an absorptive
mode of nutrition. Fungi typically contain chitin in their cell
walls and exhibit mycelial or yeast-like growth habits (More Gene
Manipulations in Fungi, edited by J. W. Bennet and L. L. Lasure,
Academic Press Inc. (1991), ISBN 0120886421). More specifically,
the terms refer to secondary metabolite producing organisms
including, without limitation, Aspergillus sp., Penicillium sp.,
Acremonium chrysogenum, Yarrowia lipolytica, Nodulisporium sp.,
Fusarium sp., Monascus sp., Claviceps sp., Trichoderma sp.,
Tolypocladium sp., Tricotheicium sp., Fusidium sp., Emericellopsis
sp., Cephalosporium sp., Cochliobolus sp., Helminthosporium sp.,
Agaricus brunescens, Ustilago maydis, Neurospora sp.,
Pestalotiopsis sp. and Phaffia rhodozyma (See, Fungal Physiology,
Chapter 9 (Secondary(Special) Metabolism), Griffin, D. H., John
Wiley & Sons, Inc.; ISBN: 0471166154).
[0039] The term "variant regulator protein" is used herein to refer
to any regulatory protein having at least one change or difference
in the amino acid sequence of the protein when compared to its
cognate, wild-type regulatory protein sequence. The term does not
include naturally occurring allelic variations of the cognate,
wild-type regulatory protein.
[0040] The term "regulator protein" is meant to refer to a protein
having a positive or negative function that modifies the production
of a secondary metabolite. The function of the protein may be at
the level of transcription, e.g., repression or activation, protein
synthesis, or transport. The regulator may alter the level of
transcription, RNA stability, translation, post-translational
modification, or cellular localization of proteins involved in
secondary metabolite synthesis and/or transport. The regulator may
also have effects on precursor metabolite pools, flux through
specific pathways and metabolite resistance.
[0041] By way of non-limiting example, certain embodiments of the
aspects of the invention relate to a regulator protein that is a
protein that contributes and/or promotes transcription of a gene
sequence, i.e., a transcription-activating protein.
"Transcription-activating" is a term used to refer to
characteristics of a protein that promote transcription. As used
herein, a transcription-activating protein would include proteins
that increase accessibility of the DNA to transcription complexes,
for example, by opening or relaxing chromatin structure, proteins
that promote the recognition and/or binding of transcription
complexes to a target gene sequence, and/or proteins that promote
transcription complex movement along the length of the template DNA
sequence.
[0042] Regulatory proteins of secondary metabolite production and
the nucleic acid sequences encoding these are known to those
skilled in the art. Non-limiting examples of regulatory proteins of
secondary metabolite synthesis include: regulator proteins of the
aflatoxin/sterigmatocystin biosynthetic cluster (Woloshuk, C. P.,
et al., Appl, Environ. Microbiol. 60:2408-2414 (1994) and Brown, D.
W., et al., Proc Natl Acad Sci U S A. 93:1418-1422 (1996));
regulator proteins of the paxilline biosynthetic cluster (Young,
C., et al., Mol, Microbiol. 39:754-764 (2001)); regulator proteins
of the cephalosporin and penicillin biosynthetic clusters (Litzka
O., et al., Antonie Van Leeuwenhoek 75:95-105 (1999); Schmitt E. K.
and Kuck U., J. Biol. Chem. 275:9348-9357 (2000); MacCabe et al.
Mol. Gen. Genet. 250:367-374 (1996); Suarez et al. Mol. Microbiol.
20:529-540 (1996); Lambert et al. Mol. Cell. Biol. 17:3966-3976
(1997); Su et al. Genetics 133:67-77 (1993); regulator proteins of
tricothecene synthesis (Trapp S. C., et al., Mol. Gen. Genet.
257:421-432 (1998); Brown D. W., et al., Fungal Genet. Biol.
32:121-133 (2001); and Matsumoto G., et al. Biosci. Biotechnol.
Biochem. 63:2001-2004 (1999)); and regulator proteins of lovastatin
synthesis (Kennedy, J., et al., Science 284:1368-1372 (1999);
Hendrickson et al., Chem. Biol. 6:429-439 (1999) Tag, A. et al.,
Mol Microbiol. 38:658-65 (2000)).
[0043] Certain embodiments of the aspects of the invention
disclosed herein relate to the lovE regulator protein, a protein
which plays a key role in the biosynthesis of lovastatin. More
particularly, certain embodiments of the aspects of the invention
relate to variant proteins of the lovE regulator protein and
methods of making the same. Such proteins are variant with respect
to the following A. terreus wild-type lovE sequences (SEQ ID NOS:91
and 92).
1TABLE 1 Amino Acid and Nucleic Acid Sequences of Wild-type lovE
Wild-type lovE Amino Acid Sequence
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrc (SEQ
ID NO:91) qqaglrcvysercpkrklrqsraadlvsadpdpclhmssppvpsqsl-
pldvseshssnts rqfldppdsydwswtsigtdeaidtdcwglsqcdggfscqlept-
lpdlpspfestvekap lppvssdiaraasaqrelfddlsavsqeleeillavtvewp-
kqeiwthpigmffnasrrl ltvlrqqaqadchqgtldeclrtknlftavhcyilnvr-
iltaiselllsqirrtqnshms plegsrsqspsrddtssssghssvdtipffsenlp-
igelfsyvdplthalfsacttlhvg vqllreneitlgvhsaqgiaasismsgepged-
iartgatnsarceeqpttpaarvlfmfl sdegafqeaksagsrgrtiaalrrcyedi-
fslarkhkhgmlrdlnnipp Wild-type lovE DNA Sequence
atggctgcagatcaaggtatattcacgaactcggtcactctctcgccagtggagggttca (SEQ
ID NO:92) cgcaccggtggaacattaccccgccgtgcattccgacgctcttgtgatcg-
gtgtcatgca caaaagatcaaatgtactggaaataaggaggttactggccgtgctcc-
ctgtcagcgttgc cagcaggctggacttcgatgcgtctacagtgagcgatgccccaa-
gcgcaagctacgccaa tccagggcagcggatctcgtctctgctgacccagatccctg-
cttgcacatgtcctcgcct ccagtgccctcacagagcttgccgctagacgtatccga-
gtcgcattcctcaaatacctcc cggcaatttcttgatccaccggacagctacgactg-
gtcgtggacctcgattggcactgac gaggctattgacactgactgctgggggctgtc-
ccaatgtgatggaggcttcagctgtcag ttagagccaacgctgccggatctaccttc-
gcccttcgagtctacggttgaaaaagctccg ttgccaccggtatcgagcgacattgc-
tcgtgcggccagtgcgcaacgagagcttttcgat gacctgtcggcggtgtcgcagga-
actggaagagatccttctggccgtgacggtagaatgg
ccgaagcaggaaatctggacccatccaatcggaatgtttttcaatgcgtcacgacggctt
cttactgtcctgcgccaacaagcgcaggccgactgccatcaaggcacactagacgaatgt
ttacggaccaagaacctctttacggcagtacactgttacatattgaatgtgcggattttg
accgccatatcggagttgctcctgtcgcaaattaggcggacccagaacagccatatgagc
ccactggaagggagtcgatcccagtcgccgagcagagacgacaccagcagcagcagcggc
cacagcagtgttgacaccatacccttctttagcgagaacctccctattggtgagctg- ttc
tcctatgttgaccccctgacacacgccctattctcggcttgcactacgttacat- gttggg
gtacaattgctgcgtgagaatgagattactctgggagtacactccgcccag- ggcattgca
gcttccatcagcatgagcggggaaccaggcgaggatatagccaggaca- ggggcgaccaat
tccgcaagatgcgaggagcagccgaccactccagcggctcgggtt- ttgttcatgttcttg
agtgatgaaggggctttccaggaggcaaagtctgctggttcc- cgaggtcgaaccatcgca
gcactgcgacgatgctatgaggatatcttttccctcgcc- cgcaaacacaaacatggcatg
ctcagagacctcaacaatattcctccatga
[0044] As used herein, the term "secondary metabolite" means a
compound, derived from primary metabolites, that is produced by an
organism, is not a primary metabolite, is not ethanol or a fusel
alcohol, and is not required for growth under standard conditions.
Secondary metabolites are derived from intermediates of many
pathways of primary metabolism. These pathways include, without
limitation, pathways for biosynthesis of amino acids, the shikimic
acid pathway for biosynthesis of aromatic amino acids, the
polyketide biosynthetic pathway from acetyl coenzyme A (CoA), the
mevalonic acid pathway from acetyl CoA, and pathways for
biosynthesis of polysaccharides and peptidopolysaccharides.
[0045] Collectively, secondary metabolism involves all primary
pathways of carbon metabolism. Particularly preferred in
embodiments of the aspects of the invention are fungal secondary
metabolites (See, Fungal Physiology, Chapter 9 (Secondary (Special)
Metabolism), Griffin, D. H., John Wiley & Sons, Inc.; ISBN:
0471166154).
[0046] "Secondary metabolite" also includes intermediate compounds
in the biosynthetic pathway for a secondary metabolite that are
dedicated to the pathway for synthesis of the secondary metabolite.
"Dedicated to the pathway for synthesis of the secondary
metabolite" means that once the intermediate is synthesized by the
cell, the cell will not convert the intermediate to a primary
metabolite. "Intermediate compounds" also include secondary
metabolite intermediate compounds which can be converted to useful
compounds by subsequent chemical conversion or subsequent
biotransformation. As such, providing improved availability of such
intermediate compounds would still lead to improved production of
the ultimate useful compound, which itself may be referred to
herein as a secondary metabolite. The yeast Saccharomyces
cerevisiae is not known to produce secondary metabolites.
[0047] The term "primary metabolite" means a natural product that
has an obvious role in the functioning of almost all organisms.
Primary metabolites include, without limitation, compounds involved
in the biosynthesis of lipids, carbohydrates, proteins, and nucleic
acids. The term "increasing the yield of the secondary metabolite"
means increasing the quantity of the secondary metabolite present
in the total fermentation broth per unit volume of fermentation
broth or culture.
[0048] As used herein, the phrase "modulate production of a
secondary metabolite" refers to a positive or negative or desirable
change in one or more of the variables or values that affect the
process or results of production of the primary or secondary
metabolites in a liquid or solid state fungal fermentation. These
positive or negative or desirable changes include, without
limitation, an increase or decrease in the amount of a primary or
secondary metabolite being produced (in absolute terms or in
quantity per unit volume of fermentation broth or per unit mass of
solid substrate); a decrease in the volume of the broth or the
mass/quantity of substrate required for the production of
sufficient quantities; a decrease in the cost of raw materials and
energy, the time of fermentor or culture run, or the amount of
waste that must be processed after a fermentor run; an increase or
decrease in the specific production of the desired metabolite (both
in total amounts and as a fraction of all metabolites and side
products made by the fungus); an increase or decrease in the
percent of the produced secondary metabolite that can be recovered
from the fermentation broth or culture; and an increase in the
resistance of an organism producing a primary or secondary
metabolite to possible deleterious effects of contact with the
secondary metabolite.
[0049] In certain embodiments of aspects of the invention, a
secondary metabolite is an anti-bacterial. An "anti-bacterial" is a
molecule that has cytocidal or cytostatic activity against some or
all bacteria. Preferred anti-bacterials include, without
limitation, .beta.-lactams. Preferred .beta.-lactams include,
without limitation, penicillins and cephalosporins and biosynthetic
intermediates thereof. Preferred penicillins and biosynthetic
intermediates include, without limitation, isopenicillin N,
6-aminopenicillanic acid (6-APA), penicillin G, penicillin N, and
penicillin V. Preferred cephalosporins and biosynthetic
intermediates include, without limitation, deacetoxycephalosporin V
(DAOC V), deacetoxycephalosporin C (DAOC), deacetylcephalosporin C
(DAC), 7-aminodeacetoxycephalosporanic acid (7-ADCA), cephalosporin
C, 7-B-(5-carboxy-5-oxopentanamido)-cephalosporanic acid
(keto-AD-7ACA), 7-B -(4-carboxybutanamido)-cephalosporanic acid
(GL-7ACA), and 7-aminocephalosporanic acid (7ACA).
[0050] In certain embodiments of aspects of the invention, the
secondary metabolite is an anti-hypercholesterolemic or a
biosynthetic intermediate thereof. An "anti-hypercholesterolemic"
is a drug administered to a patient diagnosed with elevated
cholesterol levels for the purpose of lowering the cholesterol
levels. Preferred anti-hypercholesterolemics include, without
limitation, lovastatin, mevastatin, simvastatin, and
pravastatin.
[0051] According to other embodiments of the invention, a secondary
metabolite is an immunosuppressant or a biosynthetic intermediate
thereof. An "immunosuppressant" is a molecule that reduces or
eliminates an immune response in a host when the host is challenged
with an immunogenic molecule, including immunogenic molecules
present on transplanted organs, tissues or cells. Preferred
immunosuppressants include, without limitation, members of the
cyclosporin family and beauverolide L. Preferred cyclosporins
include, without limitation, cyclosporin A and cyclosporin C.
[0052] In certain embodiments of aspects of the invention, the
secondary metabolite is an ergot alkaloid or a biosynthetic
intermediate thereof. An "ergot alkaloid" is a member of a large
family of alkaloid compounds that are most often produced in the
sclerotia of fungi of the genus Claviceps. An "alkaloid" is a small
molecule that contains nitrogen and has basic pH characteristics.
The classes of ergot alkaloids include clavine alkaloids, lysergic
acids, lysergic acid amides, and ergot peptide alkaloids. Preferred
ergot alkaloids include, without limitation, ergotamine, ergosine,
ergocristine, ergocryptine, ergocornine, ergotaminine, ergosinine,
ergocristinine, ergocryptinine, ergocorninine, ergonovine,
ergometrinine, and ergoclavine.
[0053] In certain embodiments of aspects of the invention, the
secondary metabolite is an inhibitor of angiogenesis or a
biosynthetic intermediate thereof. An "angiogenesis inhibitor" is a
molecule that decreases or prevents the formation of new blood
vessels. Angiogenesis inhibitors have proven effective in the
treatment of several human diseases including, without limitation,
cancer, rheumatoid arthritis, and diabetic retinopathy. Preferred
inhibitors of angiogenesis include, without limitation, fumagillin
and ovalicin.
[0054] In certain embodiments of aspects of the invention, the
secondary metabolite is a glucan synthase inhibitor or a
biosynthetic intermediate thereof. A "glucan synthase inhibitor" is
a molecule that decreases or inhibits the production of
1,3-.beta.-D-glucan, a structural polymer of fungal cell walls.
Glucan synthase inhibitors are a class of antifungal agents.
Preferred glucan synthase inhibitors include, without limitation,
echinocandin B, pneumocandin B, aculeacin A, and papulacandin.
[0055] In certain embodiments of aspects of the invention, the
secondary metabolite is a member of the gliotoxin family of
compounds or a biosynthetic intermediate thereof. The "gliotoxin
family of compounds" are related molecules of the
epipolythiodioxopiperazine class. Gliotoxins display diverse
biological activities, including, without limitation,
antimicrobial, antifungal, antiviral, and immunomodulating
activities. Preferred members of the "gliotoxin family of
compounds" include, without limitation, gliotoxin and
aspirochlorine.
[0056] In certain embodiments of aspects of the invention, the
secondary metabolite is a fungal toxin or a biosynthetic
intermediate thereof. A "fungal toxin" is a compound that causes a
pathological condition in a host, either plant or animal. Fungal
toxins could be mycotoxins present in food products, toxins
produced by phytopathogens, toxins from poisonous mushrooms, or
toxins produced by zoopathogens. Preferred fungal toxins include,
without limitation, aflatoxins, patulin, zearalenone, cytochalasin,
griseofulvin, ergochrome, cercosporin, marticin, xanthocillin,
coumarins, tricothecenes, fusidanes, sesterpenes, amatoxins,
malformin A, phallotoxins, pentoxin, HC toxin, psilocybin,
bufotenine, lysergic acid, sporodesmin, pulcheriminic acid,
sordarins, fumonisins, ochratoxin A, and fusaric acid.
[0057] With some certain embodiments of aspects of the invention,
the secondary metabolite is a modulator of cell surface receptor
signaling or a biosynthetic intermediate thereof. The term "cell
surface receptor" is as used before. Modulators of cell surface
receptor signaling might function by one of several mechanisms
including, without limitation, acting as agonists or antagonists,
sequestering a molecule that interacts with a receptor such as a
ligand, or stabilizing the interaction of a receptor and molecule
with which it interacts. Preferred modulators of cell surface
signaling include, without limitation, the insulin receptor agonist
L-783,281 and the cholecystokinin receptor antagonist
asperlicin.
[0058] In certain embodiments of aspects of the invention, the
secondary metabolite is a plant growth regulator or a biosynthetic
intermediate thereof. A "plant growth regulator" is a molecule that
controls growth and development of a plant by affecting processes
that include, without limitation, division, elongation, and
differentiation of cells. Preferred plant growth regulators
include, without limitation, cytokinin, auxin, gibberellin,
abscisic acid, and ethylene.
[0059] In certain embodiments of aspects of the invention, the
secondary metabolite is a pigment or a biosynthetic intermediate
thereof. A "pigment" is a substance that imparts a characteristic
color. Preferred pigments include, without limitation, melanins and
carotenoids.
[0060] In certain embodiments of aspects of the invention, the
secondary metabolite is an insecticide or a biosynthetic
intermediate thereof. An "insecticide" is a molecule that is toxic
to insects. Preferred insecticides include, without limitation,
nodulisporic acid.
[0061] In certain embodiments of aspects of the invention, the
secondary metabolite is an anti-neoplastic compound or a
biosynthetic intermediate thereof. An "anti-neoplastic" compound is
a molecule that prevents or reduces tumor formation. Preferred
anti-neoplastic compounds include, without limitation, taxol
(paclitaxel) and related taxoids.
[0062] The phrase "increased activity" is used herein to refer to a
characteristic that results in an augmentation of the inherent
negative or positive function of the regulatory protein.
[0063] The invention provides variant regulator proteins of
secondary metabolite production with increased activity and methods
of producing the same. The invention further provides for the
identification of specific amino acid residues that are important
to the functioning of secondary metabolite regulator proteins. By
way of non-limiting example, variant regulator proteins of the
secondary metabolite regulator lovE are presented herein.
[0064] As known to those skilled in the art, certain substitutions
of one amino acid for another may be tolerated at one or more amino
acid residues of a wild-type regulator protein absent a change in
the structure, activity and/or function of the wild-type protein.
Such substitutions are referred to in the art as "conservative"
substitutions, and amino acids may be categorized into groups that
identify which amino acids may be substituted for another without
altering the structure and/or function of the protein.
[0065] As used herein, the term "conservative substitution" refers
to the exchange of one amino acid for another in the same
conservative substitution grouping in a protein sequence.
Conservative amino acid substitutions are known in the art and are
generally based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. In a preferred
embodiment, conservative substitutions typically include
substitutions within the following groups: Group 1: glycine,
alanine, and proline; Group 2: valine, isoleucine, leucine, and
methionine; Group 3: aspartic acid, glutamic acid, asparagine,
glutamine; Group 4: serine, threonine, and cysteine; Group 5:
lysine, arginine, and histidine; Group 6: phenylalanine, tyrosine,
and tryptophan. Each group provides a listing of amino acids that
may be substituted in a protein sequence for any one of the other
amino acids in that particular group.
[0066] As stated supra, there are several criteria used to
establish groupings of amino acids for conservative substitution.
For example, the importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, Mol. Biol.
157:105-132 (1982). It is known that certain amino acids may be
substituted for other amino acids having a similar hydropathic
index or score and still retain a similar. biological activity.
Amino acid hydrophilicity is also used as a criteria for the
establishment of conservative amino acid groupings (see, e.g., U.S.
Pat. No. 4,554,101).
[0067] Information relating to the substitution of one. amino acid
for another is generally known in the art (see, e.g., Introduction
to Protein Architecture: The Structural Biology of Proteins, Lesk,
A. M., Oxford University Press; ISBN: 0198504748; Introduction to
Protein Structure, Branden, C.-I., Tooze, J., Karolinska Institute,
Stockholm, Sweden (January 15, 1999); and Protein Structure
Prediction: Methods and Protocols (Methods in Molecular Biology),
Webster, D. M. (Editor), August 2000, Humana Press, ISBN:
0896036375).
[0068] In one embodiment of the first aspect, the invention
provides an improved regulator protein comprising an amino acid
sequence coding for a variant of the lovE protein having at least
one specific mutation that gives rise to greater
transcription-activating properties of the regulator protein and/or
increased lovastatin synthesis.
[0069] By way of non-limiting example, certain amino acid residues
and mutations thereof in the lovE regulatory protein of A. terreus
(SEQ ID NO:91) are identified by the invention described herein.
Mutations at residues 31, 41, 52, 73, 101, 111, 133, 141, 153, 281,
367, and 389 of the wild-type lovE protein of A. terreus have been
identified as being critical for the improvement of lovE regulator
protein function. Those mutations include: F31L, Q41K, Q41R, T52I,
T52N, C73R, P101S, P101Q, V111I, S133L, E141V, E141K, C153Y, C153R,
T281A, N367I, N367Y, P389S and P389L. Each mutation, therefore,
represents a change of one conservative class of amino acids for
another. For example, the mutation F31L represents a change from a
Group 6 amino acid residue to a Group 2 amino acid residue at
position 31 of the wild-type, lovE regulator protein.
[0070] Thus, by way of non-limiting example, regulator proteins of
this aspect of the invention include at least one of the following
mutations: (1) a Group 6 amino acid residue mutated to a Group 2
amino acid residue at position 31, for example, the mutation
represented by F31L; (2) a Group 3 amino acid residue mutated to a
Group 5 amino acid residue at position 41, for example, the
mutation represented by Q41K or Q41R; (3) a Group 4 amino acid
residue mutated to a Group 2 amino acid residue at position 52, for
example, the mutation represented by T52I; (4) a Group 4 amino acid
residue mutated to a Group 3 amino acid residue at position 52, for
example, the mutation represented by T52N; (5) a Group 4 amino acid
residue mutated to a Group 5 amino acid residue at position 73, for
example, the mutation represented by C73R; (6) a Group 1 amino acid
residue mutated to a Group 4 amino acid residue at position 101,
for example, the mutation represented by P101S; (7) a Group 1 amino
acid residue mutated to a Group 3 amino acid residue at position
101, for example, the mutation represented by P101Q; (8) a valine
amino acid residue mutated to another Group 2 amino acid residue at
position 111, for example, the mutation represented by V111I; (9) a
Group 4 amino acid residue mutated to a Group 2 amino acid residue
at position 133, for example, the mutation represented by S133L;
(10) a Group 3 amino acid residue mutated to a Group 2 amino acid
residue at position 141, for example, the mutation represented by
E141V; (11) a Group 3 amino acid residue mutated to a Group 5 amino
acid residue at position 141, for example, the mutation represented
by E141K; (12) a Group 4 amino acid residue mutated to Group 6
amino acid residue at position 153, for example, the mutation
represented by C153Y; (13) a Group 4 amino acid residue mutated to
a Group 5 amino acid residue at position 153, for example, the
mutation represented by C153R; (14) a Group 4 amino acid residue
mutated to a Group 1 amino acid residue at position 281, for
example, the mutation represented by T281A; (15) a Group 3 amino
acid residue mutated to a Group 2 amino acid residue at position
367, for example, the mutation represented by N367I; (16) a Group 3
amino acid residue mutated to a Group 6 amino acid residue at
position 367, for example, the mutation represented by N367Y; (17)
a Group 1 amino acid residue mutated to Group 4 amino acid residue
at position 389, for example, the mutation represented by P389S;
and/or (18) a Group 1 amino acid residue mutated to a Group 2 amino
acid residue at position 389, for example, the mutation represented
by P389L.
[0071] In other embodiments of the first aspect, the invention
provides a variant of the lovE regulator protein with at least two,
or at least three, or at least four, or at least five, or at least
six, or at least seven, or at least eight, or at least nine, or at
least ten, or at least eleven, or at least twelve, or at least
thirteen, or at least fourteen, or at least fifteen, or at least
sixteen, or at least seventeen, or at least eighteen of the above
described specific mutations.
[0072] In other embodiments of the first aspect, the invention
provides an isolated lovE variant regulator protein having the
sequence of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,
SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID
NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ
ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,
SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID
NO:63, SEQ ID NO:64, or SEQ ID NO:65.
[0073] In a second aspect, the invention provides a nucleic acid
molecule encoding a variant regulator protein of secondary
metabolite production of the first aspect of the invention. As used
herein, the terms "nucleic acid" or "nucleic acid molecule" refer
to a deoxyribonucleotide or ribonucleotide polymer in either
single- or double-stranded form, and unless otherwise limited,
would encompass analogs of natural nucleotides that can function in
a similar manner as the naturally occurring nucleotide.
[0074] In one embodiment of the second aspect, the invention
provides a nucleic acid molecule encoding a variant protein of the
lovE regulator protein of the first aspect of the invention.
[0075] By way of non-limiting example, the invention provides a
nucleic acid molecule encoding a lovE variant regulator protein
having the sequence of SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,
SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,
SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or SEQ ID
NO:90.
[0076] Poor transformation efficiency and the lack of efficient
selection systems frequently precludes the screening of large
numbers of variant regulator proteins of secondary metabolites in
the organism from which the regulator protein is isolated. For
example, there are currently certain technical obstacles to the
successful screening of large numbers of variant regulator proteins
in the fungus A. terreus, an organism that produces the secondary
metabolite lovastatin.
[0077] The invention described herein takes advantage of the
genetically tractable and experimentally amenable organism
Saccharomyces cerevisiae for screening large numbers of variant
regulator proteins of secondary metabolite production. Techniques
common to the field of molecular biology are well developed in S.
cerevisiae, and large numbers of vectors are available to assist
the genetic manipulation and cloning of variant regulator proteins
involved in secondary metabolite production. Other genetically
tractable organisms could also be used for this purpose.
[0078] In a third aspect, the invention provides a method of
increasing the activity of a protein that regulates secondary
metabolite production comprising: (a) selecting a nucleic acid
comprising a polynucleotide encoding a protein regulator of
secondary metabolite production; (b) mutating the nucleic acid to
create a plurality of nucleic acid molecules encoding variant
regulator proteins of secondary metabolite production; and (c)
selecting a variant regulator protein with more activity than the
cognate, wild-type protein.
[0079] As used herein, "mutating" is used to refer to the
deliberate alteration of at least one nucleotide residue of a
wild-type, cognate nucleic acid sequence encoding a regulator
protein of secondary metabolite production. A deliberate alteration
or change in at least one nucleotide residue of a polynucleotide
may be accomplished by any method known in the art. The mutation(s)
can be made in vivo or in vitro and can include random, partially
random or not random, i.e., directed, mutagenesis techniques.
[0080] By way of non-limiting example, in vivo mutagenesis can be
done by placing this nucleic acid molecule in a cell with a high
mutation frequency, i.e. a mutagenic strain. By way of non-limiting
example, Muhlrad et al. (Yeast 8:79-82 (1992)) have developed a
rapid method for localized mutagenesis of yeast genes. As a first
step, the region of interest of a gene sequence is first amplified
in vitro under error-prone polymerase chain reaction (PCR)
conditions. Error-prone polymerase chain reaction (PCR) is a method
of introducing amino acid changes into proteins. With this
technique, mutations are deliberately introduced during the PCR
reaction through the use of error-prone DNA polymerases under
specific reaction conditions. With the Muhlrad et al. procedure,
the PCR product is then co-transformed with a gapped plasmid
containing homology to both ends of the PCR product, resulting in
in vivo recombination to repair the gap with the mutagenized
DNA.
[0081] There are a variety of commercially available kits that may
be used to produce mutant nucleic acid molecules by error-prone PCR
(see, e.g., GeneMorph.TM. PCR Mutagenesis Kit (Stratagene, La
Jolla, Calif.); and Diversify.TM. PCR Random Mutagenesis Kit (BD
Biosciences Clontech, Palo Alto, Calif.). Thus, a plurality of
variant, i.e., mutated, regulator proteins of secondary metabolite
production may be produced using established mutagenesis
techniques.
[0082] As used herein, the term "activity" refers to a
characteristic of the regulator protein that negatively or
positively affects the biological system to bring about a
modulation in secondary metabolite production. By way of
non-limiting example, the activity is the transcription of
downstream genes involved in the biosynthetic pathway of the
secondary metabolite of choice. Thus, in the present example, the
phrase "more activity" refers to the property of a variant
regulator protein to bring about more transcription than that
effected by the cognate, wild-type regulator protein.
[0083] In certain embodiments of the third aspect, the selected
variant regulator protein has more activity in a fungal cell than
the cognate, wild-type protein. In certain embodiments of the third
aspect, the protein regulator of secondary metabolite production is
a transcription factor. In certain embodiments of the fourth
aspect, the protein regulator of secondary metabolite production is
a transmembrane transporter, a protein that mediates secretion, a
kinase, a G-protein, a cell surface receptor, a GTPase activating
protein, a guanine nucleotide exchange factor, a phosphatase, a
protease, a phosphodiesterase, a bacterial protein toxin, an
importin, an RNA-binding protein, an SCF complex component, an
adherin, or a protein encoded within a biosynthetic cluster. . In
certain other embodiments of the third aspect, the selected variant
regulator protein has more activity in a heterologous cell than the
cognate, wild-type protein. In certain embodiments thereof, the
heterologous cell is an organism selected from the group consisting
of S. cerevisiae, E. coli, A. nidulans, Candida sp., and N. crassa.
In yet certain other embodiments of the third aspect, the selected
variant regulator protein has more activity in a homologous cell
than the cognate, wild-type protein. In certain embodiments
thereof, the homologous cell is an organism selected from the group
consisting of Aspergillus sp., Penicillium sp., Acremonium
chrysogenum, Yarrowia lipolytica, Nodulisporium sp., Fusarium sp.,
Monascus sp., Claviceps sp., Trichoderma sp., Tolypocladium sp.,
Tricotheicium sp., Fusidium sp., Emericellopsis sp., Cephalosporium
sp., Cochliobolus sp., Helminthosporium sp., Agaricus brunescens,
Ustilago maydis, Neurospora sp., Pestalotiopsis sp., and Phaffia
rhodozyma.
[0084] In certain embodiments of the third aspect, the selected
variant regulator protein has more activity in a heterologous cell
and a homologous cell than the cognate, wild-type protein. In
certain embodiments thereof, the heterologous cell is an organism
selected from the group consisting of S. cerevisiae, E. coli, A.
nidulans, Candida sp., and N. crassa and the homologous cell is an
organism selected from the group consisting of Aspergillus sp.,
Penicillium sp., Acremonium chrysogenum, Yarrowia lipolytica,
Nodulisporium sp., Fusarium sp., Monascus sp., Claviceps sp.,
Trichoderma sp., Tolypocladium sp., Tricotheicium sp., Fusidium
sp., Emericellopsis sp., Cephalosporium sp., Cochliobolus sp.,
Helminthosporium sp., Agaricus brunescens, Ustilago maydis,
Neurospora sp., Pestalotiopsis sp. and Phaffia rhodozyma.
[0085] As used herein, the phrase "heterologous cell" refers to a
system for gene expression, i.e., an organism for gene expression,
that is one other than the organism from which the selected
regulator protein of secondary metabolite production has been
isolated. Preferred heterologous cells include, but are not limited
to, S. cerevisiae, E. coli, A. nidulans, and Candida sp., . and N.
crassa. Particularly preferred are fungal heterologous cells. In an
embodiment of the third aspect, the method comprises: (a) selecting
a nucleic acid comprising a polynucleotide encoding a protein
regulator of secondary metabolite production; (b) mutating the
nucleic acid to create a plurality of nucleic acid molecules
encoding variant regulator proteins of secondary metabolite
production; and (c) selecting a mutagenized nucleic acid encoding a
variant regulator protein with increased activity in a homologous
cell than the cognate, wild-type protein.
[0086] As used herein, the phrase "homologous cell" refers to a
system for gene expression, i.e., an organism for gene expression,
that is the organism from which the regulator protein of secondary
metabolite production has been isolated. Preferred homologous cells
are fungal homologous cells, including, but not limited to,
Aspergillus sp., Penicillium sp., Acremonium chrysogenum, Yarrowia
lipolytica, Nodulisporium sp., Fusarium sp., Monascus sp.,
Claviceps sp., Trichoderma sp., Tolypocladium sp., Tricotheicium
sp., Fusidium sp., Emericellopsis sp., Cephalosporium sp.,
Cochliobolus sp., Helminthosporium sp., Agaricus brunescens,
Ustilago maydis, Neurospora sp., Pestalotiopsis sp and Phaffia
rhodozyma. (See, Fungal Physiology, Chapter 9 (Secondary (Special)
Metabolism), Griffin, D. H., John Wiley & Sons, Inc.; ISBN:
0471166154).
[0087] In certain embodiments of the third aspect, the method
further comprises selecting a variant regulator protein that also
increases production of a secondary metabolite in a cell when
compared to the cognate, wild-type protein. In certain embodiments
thereof, the cell is a fungal cell. In certain embodiments thereof,
the cell is a heterologous cell, preferably selected from the group
consisting of S. cerevisiae, E. coli, A. nidulans, Candida sp., and
N. crassa.
[0088] In certain embodiments thereof, the cell is a homologous
cell, preferably selected from the group consisting of Aspergillus
sp., Penicillium sp., Acremonium chrysogenum, Yarrowia lipolytica,
Nodulisporium sp., Fusarium sp., Monascus sp., Claviceps sp.,
Trichoderma sp., Tolypocladium sp., Tricotheicium sp., Fusidium
sp., Emericellopsis sp., Cephalosporium sp., Cochliobolus sp.,
Helminthosporium sp., Agaricus brunescens, Ustilago maydis,
Neurospora sp., Pestalotiopsis sp., and Phaffia rhodozyma.
[0089] Certain embodiments of the aspects of the invention relate
to regulator proteins that promote secondary metabolite production
by increasing transcription of one or more genes involved with
secondary metabolite production. These wild-type sequences may be
selected for mutagenesis to create a plurality of variant regulator
proteins. The activity of these transcription-activating variant
regulator proteins may be determined by measuring the activity of a
reporter gene having the appropriate promoter sequences. These
tests are done in a homologous and/or a heterologous cell. Certain
embodiments of aspects of the invention are directed to fungal
regulator proteins with transcription-activating activity that is
tested in fungal heterologous and homologous cells.
[0090] Reporter genes are useful for isolating transformants
expressing improved variant regulator proteins. The reporter genes
may be operably linked to a promoter sequence that is normally
regulated by the wild-type regulator protein. Reporter genes
include, but are not limited to, genes encoding
.beta.-galactosidase (lacZ), .beta.-glucoronidase (GUS),
.beta.-glucosidase, amylase and invertase, amino acid biosynthetic
genes, e.g., the yeast LEU2, HIS3, LYS2, TRP1 genes (or homologous
genes from other fungi, such as filamentous fungi, that encode
proteins with the similar functional activities), nucleic acid
biosynthetic genes, e.g., the yeast URA3 and ADE2 genes (or
homologous genes from other fungi, such as filamentous fungi, that
encode proteins with the similar functional activities), the
mammalian chloramphenicol transacetylase (CAT) gene, or any surface
antigen gene for which specific antibodies are available. A
reporter gene can also be a neomycin phosphotransferase(neo) gene,
which encodes neomycin, kanamycin resistance gene and G418
(geneticin) resistance gene. A reporter gene may encode a protein
detectable by luminescence or fluorescence, such as green
fluorescent protein (GFP). Reporter genes may additionally or
alternatively encode any protein that provides a phenotypic marker,
for example, a protein that is necessary for cell growth or
viability, or a toxic protein that causes cell death.
Alternatively, the reporter gene may encode a protein detectable by
a color assay leading to the presence or absence of color.
[0091] The choice of reporter gene will depend on the type of cell
to be transformed. Preferred reporter genes are those that are
operable in fungal cells. It is preferable to have two reporter
genes within the cell. One reporter gene, when expressed, provides
a growth advantage to transformed cells that are expressing the
variant regulator protein. This allows for the isolation of such
transformants though selective pressures. The other reporter gene
provides a calorimetric marker, such as the lacZ gene and its
encoded protein, .beta.-galactosidase. Alternatively, the second
reporter provides a fluorescent or luminescent marker, such as
green fluorescent protein (GFP).
[0092] In a fourth aspect, the invention provides a method of
increasing production of a secondary metabolite comprising: (a)
selecting a nucleic acid comprising a polynucleotide encoding a
protein regulator of secondary metabolite production; (b) mutating
the nucleic acid to create a plurality of nucleic acid molecules
encoding variant regulator proteins of secondary metabolite
production; (c) selecting a variant regulator protein with more
activity than the cognate, wild-type protein; and (d) expressing
the selected variant regulator protein in a cell, thereby
increasing production of the secondary metabolite in the cell.
[0093] In certain embodiments of the fourth aspect, the cell is a
fungal cell. In certain embodiments of the fourth aspect, the
protein regulator of secondary metabolite production is a
transcription factor. In certain embodiments of the fourth aspect,
the protein regulator of secondary metabolite production is a
transmembrane transporter, a protein that mediates secretion, a
kinase, a G-protein, a cell surface receptor, a GTPase activating
protein, a guanine nucleotide exchange factor, a phosphatase, a
protease, a phosphodiesterase, a bacterial protein toxin, an
importin, an RNA-binding protein, an SCF complex component, an
adherin, or a protein encoded within a biosynthetic cluster. In
certain embodiments of the fourth aspect, the cell is a
heterologous cell, preferably selected from the group consisting of
S. cerevisiae, E. coli, A. nidulans, Candida sp., and N. crassa. In
certain other embodiments of the fourth aspect, the cell is a
homologous cell, preferably selected from the group consisting of
Aspergillus sp., Penicillium sp., Acremonium chrysogenum, Yarrowia
lipolytica, Nodulisporium sp., Fusarium sp., Monascus sp.,
Claviceps sp., Trichoderma sp., Tolypocladium sp., Tricotheicium
sp., Fusidium sp., Emericellopsis sp., Cephalosporium sp.,
Cochliobolus sp., Helminthosporium sp., Agaricus brunescens,
Ustilago maydis, Neurospora sp., Pestalotiopsis sp., and Phaffia
rhodozyma.
[0094] In certain other embodiments of the fourth aspect, the cell
is a heterologous cell and the method further comprises expressing
the variant regulator protein in a homologous cell, thereby
increasing secondary metabolite production in the homologous cell.
In certain embodiments thereof, the heterologous cell is an
organism selected from the group consisting of S. cerevisiae, E.
coli, A. nidulans, Candida sp., , and N. crassa and the homologous
cell is an organism selected from the group consisting of
Aspergillus sp., Penicillium sp., Acremonium chrysogenum, Yarrowia
lipolytica, Nodulisporium sp., Fusarium sp., Monascus sp.,
Claviceps sp., Trichoderma sp., Tolypocladium sp., Tricotheicium
sp., Fusidium sp., Emericellopsis sp., Cephalosporium sp.,
Cochliobolus sp., Helminthosporium sp., Agaricus brunescens,
Ustilago maydis, Neurospora sp., Pestalotiopsis sp. and Phaffia
rhodozyma.
[0095] In a fifth aspect, the invention provides an isolated
variant regulator protein of secondary metabolite production having
increased activity compared to a cognate, wild-type protein, made
by the process comprising: (a) selecting a nucleic acid comprising
a polynucleotide encoding a protein regulator of secondary
metabolite production; (b) mutating the nucleic acid to create a
plurality of nucleic acid molecules encoding variant regulator
proteins of secondary metabolite production; (c) selecting a
variant regulator protein with more activity than the cognate,
wild-type protein; and (d) recovering the selected variant
regulator protein.
[0096] In certain embodiments of the fifth aspect, the variant
regulator protein selected has more activity in a fungal cell. In
certain embodiments of the fifth aspect, the protein regulator of
secondary metabolite production is a transcription factor. In
certain embodiments of the fifth aspect, the protein regulator of
secondary metabolite production is a transmembrane transporter, a
protein that mediates secretion, a kinase, a G-protein, a cell
surface receptor, a GTPase activating protein, a guanine nucleotide
exchange factor, a phosphatase, a protease, a phosphodiesterase, a
bacterial protein toxin, an importin, an RNA-binding protein, an
SCF complex component, an adherin, or a protein encoded within a
biosynthetic cluster. In certain embodiments of the fifth aspect,
the variant regulator protein selected has more activity in a
heterologous cell, preferably selected from the group consisting of
S. cerevisiae, E. coli, A. nidulans, Candida sp., Neurospora sp.,
Pestalotiopsis sp., and N. crassa. In certain embodiments of the
fifth aspect, the variant regulator protein selected has more
activity in a homologous cell, preferably selected from the group
consisting of Aspergillus sp., Penicillium sp., Acremonium
chrysogenum, Yarrowia lipolytica, Nodulisporium sp., Fusarium sp.,
Monascus sp., Claviceps sp., Trichoderma sp., Tolypocladium sp.,
Tricotheicium sp., Fusidium sp., Emericellopsis sp., Cephalosporium
sp., Cochliobolus sp., Helminthosporium sp., Agaricus brunescens,
Ustilago maydis, Neurospora sp., Pestalotiopsis sp., and Phaffia
rhodozyma.
[0097] In certain embodiments of the fifth aspect, the variant
regulator protein selected has more activity in a homologous cell
and a heterologous cell. In embodiments thereof, the heterologous
cell is an organism selected from the group consisting of S.
cerevisiae, E. coli, A. nidulans, Candida sp., Neurospora sp.,
Pestalotiopsis sp., and N. crassa and the homologous cell is an
organism selected from the group consisting of Aspergillus sp.,
Penicillium sp., Acremonium chrysogenum, Yarrowia lipolytica,
Nodulisporium sp., Fusarium sp., Monascus sp., Claviceps sp.,
Trichoderma sp., Tolypocladium sp., Tricotheicium sp., Fusidium
sp., Emericellopsis sp., Cephalosporium sp., Cochliobolus sp.,
Helminthosporlum sp., Agaricus brunescens, Ustilago maydis,
Neurospora sp., Pestalotiopsis sp., and Phaffia rhodozyma.
[0098] In yet another embodiment of the fifth aspect, the variant
regulator protein is a variant protein of the lovE protein having
at least one of the following mutations: (1) a Group 6 amino acid
residue mutated to a Group 2 amino acid residue at position 31, for
example, the mutation represented by F31L; (2) a Group 3 amino acid
residue mutated to a Group 5 amino acid residue at position 41, for
example, the mutation represented by Q41K or Q41R; (3) a Group 4
amino acid residue mutated to a Group 2 amino acid residue at
position 52, for example, the mutation represented by T52I; (4) a
Group 4 amino acid residue mutated to a Group 3 amino acid residue
at position 52, for example, the mutation represented by T52N; (5)
a Group 4 amino acid residue mutated to a Group 5 amino acid
residue at position 73, for example, the mutation represented by
C73R; (6) a Group 1 amino acid residue mutated to a Group 4 amino
acid residue at position 101, for example, the mutation represented
by P101S; (7) a Group 1 amino acid residue mutated to a Group 3
amino acid residue at position 101, for example, the mutation
represented by P101Q; (8) a valine amino acid residue mutated to
another Group 2 amino acid residue at position 111, for example,
the mutation represented by V111I; (9) a Group 4 amino acid residue
mutated to a Group 2 amino acid residue at position 133, for
example, the mutation represented by S133L; (10) a Group 3 amino
acid residue mutated to a Group 2 amino acid residue at position
141, for example, the mutation represented by E141V; (11) a Group 3
amino acid residue mutated to a Group 5 amino acid residue at
position 141, for example, the mutation represented by E141K; (12)
a Group 4 amino acid residue mutated to Group 6 amino acid residue
at position 153, for example, the mutation represented by C153Y;
(13) a Group 4 amino acid residue mutated to a Group 5 amino acid
residue at position 153, for example, the mutation represented by
C153R; (14) a Group 4 amino acid residue mutated to a Group 1 amino
acid residue at position 281, for example, the mutation represented
by T281A; (15) a Group 3 amino acid residue mutated to a Group 2
amino acid residue at position 367, for example, the mutation
represented by N367I; (16) a Group 3 amino acid residue mutated to
a Group 6 amino acid residue at position 367, for example, the
mutation represented by N367Y; (17) a Group 1 amino acid residue
mutated to Group 4 amino acid residue at position 389, for example,
the mutation represented by P389S; and/or (18) a Group 1 amino acid
residue mutated to a Group 2 amino acid residue at position 389,
for example, the mutation represented by P389L.
[0099] In certain embodiments of this aspect of the invention, the
variant protein of the lovE protein sequence has an amino acid
sequence of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,
SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID
NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ
ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,
SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID
NO:63, SEQ ID NO:64, or SEQ ID NO:65.
[0100] In another embodiment thereof, the variant protein of the
lovE protein is encoded by a nucleic acid molecule having a
polynucleotide sequence of SEQ ID NO:66, SEQ ID NO:67, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ
ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, or
SEQ ID NO:90.
[0101] In a sixth aspect, the invention provides a fungus having
improved lovastatin production made by the process of transforming
a fungal cell with a nucleic acid molecule encoding a variant of
the lovE protein of the first aspect of the invention. In an
embodiment thereof, the nucleic acid molecule is selected from a
nucleic acid molecule of the second aspect of the invention.
[0102] In a seventh aspect, the invention provides an improved
process for making lovastatin comprising transforming a fungal cell
with a nucleic acid molecule encoding a variant of the lovE protein
of the first aspect of the invention. In an embodiment thereof, the
fungal cell is transformed with a nucleic acid molecule of the
second aspect of the invention.
[0103] International Patent Application PCT/US99/29583 discloses
lovastatin production genes. However, this reference does not
provide a mature lovE cDNA sequence. The invention herein remedies
the shortcoming of this reference by providing a complete cDNA
sequence for the lovE mRNA.
[0104] In an eighth aspect, the invention provides a nucleic acid
molecule encoding a lovE protein defined by SEQ ID NO:91. In an
embodiment thereof, the invention provides an isolated lovE nucleic
acid molecule defined by SEQ ID NO:92.
[0105] The following examples illustrate the preferred modes of
making and practicing the present invention but are not meant to
limit the scope of the invention since alternative methods may be
utilized to obtain similar results.
EXAMPLES
Example 1
[0106] Preparation of Strains and Plasmids
[0107] Strain MY2124 was derived from the Sigma 1278b strain
background of S. cerevisiae and its complete genotype is as
follows: MAT.alpha./MAT.alpha.::LEU2 ura3.DELTA.0/ura3.DELTA.0
leu2.DELTA.0/leu2.DELTA.0 trp1.DELTA.0::hisG/trp1.DELTA.0::hisG
his3.DELTA.0::hisG/his3.DELTA.0::hisG
ura3.DELTA.0::lovF-HIS3p-neo/ura3.D- ELTA.0. MY2124 can be
constructed by mating S. cerevisiae strains MY2112 (MAT.alpha.
ura3.DELTA.0 leu2.DELTA.0 trp1.DELTA.0::hisG his3.DELTA.0::hisG
ura3.DELTA.0::lovFp-HIS3p-neo) with MY1555 (mat.alpha.::LEU2
ura3.DELTA.0 leu2.DELTA.0 trp1.DELTA.0::hisG his3.DELTA.0::hisG)
and isolating zygotes. The ura3.DELTA.0::lovFp-HIS3p-- neo allele
of MY2112 was derived by cotransforming SfiI-linearized plasmid
MB2254 with pRS424 (Sikorski and Hieter (1989) Genetics 122:19-27)
into MY1413 (MAT.alpha. leu2.DELTA.0 trp1.DELTA.0::hisG
his3.DELTA.0::hisG). Transformants were selected on SC-Trp media
and subsequently screened for 5-fluoro-orotic acid resistance to
identify those transformants containing the
ura3.DELTA.0::lovFp-HIS3p-neo allele. Trp segregants lacking
plasmid pRS424 were isolated by growing the strain under
non-selective conditions.
[0108] The following oligonucleotides were used in the construction
of plasmids.
2TABLE 2 Oligonucleotides Utilized For LovE Variant Cloning MO664
(5'GGCCATGGAGGCCGCTAGCTCGAGTCGACGGCC- TAGGTGGCCAGCT3') (SEQ ID
NO:1) MO665 (5'GGCCACCTAGGCCGTCGACTCGAGCTAGCGGCCTCCATGGCCGTAC3')
(SEQ ID NO:2) MO666 (5'GGCGGCCGCTCTAGAACTAGTCTCGAGGGTACC3') (SEQ ID
NO:3) MO667 (5'GGTACCCTCGAGACTAGTTCTAGAGCGGCCGCC3') (SEQ ID NO:4)
MO1794 (5'CACAGCGGCCGCTCAACCTTCCCATTGGGGC3') (SEQ ID NO:5) MO1793
(5'CACCACTAGTACGCGGGCTGATTCGAC3') (SEQ ID NO:6) MO1785
(5'CACCACTAGTTATACATTATATAAAGTAATGTG3') (SEQ ID NO:7) MO1786
(5'CACAGGATCCGTCATCTTTGCCTTCGTTTATC3') (SEQ ID NO:8) MO195
(5'CGCGGATCCTATTGAACAAGATGGATTGCAC3') (SEQ ID NO:9) MO196
(5'CCGGAATTCAGAAGAACTCGTCAAGAAG3') (SEQ ID NO:10) MO841
(5'ACAAAAAAGCAGGCTCCACAATGGCTGCAGATCAAGGTAT3') (SEQ ID NO:11) MO842
(5'ACAAGAAAGCTGGGTTCATGGAGGAATATTGTTGA3') (SEQ ID NO:12) MO2278
(5'GGGGATCCAATCGAGGTCCACGACCAGT3') (SEQ ID NO:13) MO343
(5'GGGGACAAGTTTGTACAAAAAAGCAGGCT3') (SEQ ID NO:14) MO2273
(5'GGGGATCCGCCAATGGTCCCGTTCAAAC3') (SEQ ID NO:15) MO2274
(5'ACAAGAAAGCTGGGTTCACAGAATGTTTAGCT- CAA3') (SEQ ID NO:16) MO344
(5'GGGGACCACTTTGTACAAGAAAGCTGG- GT3') (SEQ ID NO:17) MO2624
(5'GCGATGCCCCAAGCGCAAGCTACGCCA- ATCCAGGG3') (SEQ ID NO:18) MO2654
(5'CGTCGCGCCATTCGCCATTCA- GGCTGCGCAACTGT3') (SEQ ID NO:19) MO2680
(5'GGACCTTTGCAGCATAAATTACTATACTTCT3') (SEQ ID NO:20) MO2686
(5'GGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGT3') (SEQ ID NO:21) MO2681
(5'TAAAACTCTTGTTTTCTTCTTTTCTCTAAAT3') (SEQ ID NO:22) MO2700
(5'CAGTGAGCGCGCGTAATACGACTCACTATAGGGCGA3') (SEQ ID NO:23) MO2701
(5'ATACTTCTATAGACACACAAACACAAATACACACAC3') (SEQ ID NO:24) MO107
(5'CGCGGATCCCGTCGTTTTACAAC3') (SEQ ID NO:25) MO197
(5'CCCAAGCTTATTATTTTTGACACCAGACCAA3') (SEQ ID NO:26) MO1293
(5'GGAAGATCTAGCATCGTGGCCAATTTCTTCTAGTTT3') (SEQ ID NO:27) MO1294
(5'ATAAGAATGCGGCCGCTCAACCTTCCCATTGG- GGCGTTTGC3') (SEQ ID NO:28)
MO1787 (5'CACAGGATCCAGCATTATTA- ATTTAGTGTGTGTATTT3') (SEQ ID NO:29)
MO1788 (5'CACCACTAGTCTCGAGCAGATCCGCCAG3') (SEQ ID NO:30) MO1793
(5'CACCACTAGTACGCGGGCTGATTCGAC3') (SEQ ID NO:31) MO1794
(5'CACAGCGGCCGCTCAACCTTCCCATTGGGGC3') (SEQ ID NO:32) MO511
(5'GGCCATCGATACAAGTTTGTACAAAAAAGCTGAAC3') (SEQ ID NO:33) MO540
(5'GGCGCCCTATTACACCACTTTGTACAAGAAAGC3') (SEQ ID NO:34) MO1985
(5'CACACGTCTCCGGCCTCAACCTTCCCATTGGGGCG3') (SEQ ID NO:35) MO1986
(5'CACACAGATCTCGTGGCCAATTTCTTCTAGTTTGA3') (SEQ ID NO:36) MO1992
(5'CACACGGATCCACAATGTTACGTCCTGTAGAAACCCC3') (SEQ ID NO:37) MO1993
(5'CACAGCGGCCGCTTCATTGTTTGCCTCCCTGC- TG3') (SEQ ID NO:38) MO316
(5'GCGGCCGCGGCGCCCGGCCCATGTCAAC- AAGAAT3') (SEQ ID NO:39) MO318
(5'CCGCGGCCGAGTGGAGATGTGGAG- T3') (SEQ ID NO:40)
[0109] Plasmid MB2254 contains the lovFp-HIS3p-neo reporter gene
flanked by URA3 sequence. First primers M0664 (SEQ ID NO:1) and
M0665 (SEQ ID NO:2) were annealed and inserted into the KpnI-SacI
sites of plasmid pBluescript II KS (Stratagene,). The resulting
vector, MB1038, contains a SalI site in the polylinker. Next, the
SpeI-XhoI fragment from pJL164 (Brachmann et al. Yeast 14:115-132
(1998)) containing a deletion of the URA3 gene with additional
flanking sequences was inserted into the NheI-SalI sites of MB1038
to create MB1053. Primers MO666 (SEQ ID NO:3) and MO667 (SEQ ID
NO:4) that contain multiple restriction sites (NotI, XbaI, SpeI,
XhoI and KpnI) were then annealed together and ligated into the
SmaI site of MB1053 to create MB1054. Next, the following four
fragments were combined in MB1054 to obtain plasmid MB2254. The
lovF promoter from A. terreus genomic DNA was PCR amplified with
MO1794 (SEQ ID NO:5) and MO1793 (SEQ ID NO:6) and inserted into
MB1054 on a NotI-SpeI fragment. The HIS3 basal promoter from pRS403
(Sikorski and Hieter, Genetics 122:19-27 (1989)) was PCR amplified
with primers MO1785 (SEQ ID NO:7) and MO1786 (SEQ ID NO:8) and
inserted into MB1054 on a SpeI-BamHI fragment. Finally, the neo
gene (PCR amplified with MO195 (BamHI) (SEQ ID NO:) and MO196
(EcoRI) (SEQ ID NO:10) from plasmid pYX11 (Xiao and Weaver, Nucl.
Acids Res. 25:2985-2991 (1997)) and CYC1 terminator sequences
(XhoI-KpnI fragment from pRS426-GAL-S (Mumberg, et al., Nucl.
Acids. Res. 22:5767-5768 (1994)) were first combined in pRS416
(Sikorski and Hieter, Genetics 122:19-27 (1989)) and then cut out
with BamHI-KpnI and inserted into MB1054 to create MB2254.
[0110] The lovFp-HIS3p-neo reporter in MY2124 can confer resistance
to the drug geneticin (G418). It was empirically determined that
MY2124 (untransformed or transformed with parental plasmids MB2478
(CYC1-lovE/CEN) or MB2848 (CYC1-lovE/At274/CEN) was unable to grow
on YPD media supplemented with 100 .mu.g /ml G418. Plasmid MB2478
contains the CYC1 promoter operationally linked to the entire A.
terreus lovE open reading frame. The CYC1 promoter is a relatively
weak promoter and thus the lovE ORF in MB2478 was expressed at low
levels. MB2478 was the parental vector plasmid for creating full
length lovE variants. Plasmid MB2848 contains the CYC1 promoter
operationally linked to a chimeric open reading frame consisting of
the A. terreus lovE DNA binding domain fused to the
carboxy-terminal portion of the At274 gene (U.S. Ser. No.
60/257,431, filed Dec. 22, 2000).
[0111] MB2848 was used to create lovE variants in which the DNA
binding domain was not mutated. Both MB2478 and MB2848 contain
yeast CEN and autonomously replicating sequences and both are
maintained at 1-2 copies per cell. In contrast to strains
transformed with MB2478 or MB2848, strains transformed with plasmid
MB1644 (TEF1-lovE/2 micron) were able to grow on G418-supplemented
YPD media. The lovE gene of MB1644 is under control of the
constitutively strong S. cerevisiae TEF1 promoter. MB1644 contains
a 2-micron origin for high-copy replication in yeast. An objective
of these studies was to identify lovE variants which when expressed
at low levels could confer G418 resistance similar to the highly
expressed wild-type lovE molecule of MB1644. S. cerevisiae
expression vectors used in these studies were constructed as
follows.
[0112] MB968 is a low copy S. cerevisiae URA3 based expression
vector. MB968 was created by inserting the EcoRV fragment
(containing the destination cassette) from gateway pEZC7201
(Invitrogen.TM., Carlsbad, Calif.) into XhoI/SalI (filled in with
Klenow) linearized pRS416 CYC1 (Mumberg, et al., Gene 156:119-122
(1995)).
[0113] MB1644 and MB2478 are URA3-based S. cerevisiae expression
plasmids that contain the wild-type lovE gene. They are both
derivatives of MB1199. MB1199 was created by using primers MO841
(SEQ ID NO:11) and MO842 (SEQ ID NO:12) to amplify the lovE ORF
from A. terreus cDNA. Gateway (Invitrogen.TM., Carlsbad, Calif.)
Cloning Technology (U.S. Pat. No. 5,888,732) was used to clone the
lovE PCR fragment into the gateway entry vector pDONR206
(Invitrogen.TM., Carlsbad, Calif.) to create MB1199. Similarly,
Gateway Cloning Technology was used to transfer the lovE ORF from
MB1199 into MB968 to create MB2478 and into MB969 (U.S. Ser. No.
60/198,335, filed Apr. 18, 2000) to create MB1644.
[0114] MB2848 is a derivative of MB968 that contains a lovE-AT274
chimera. The lovE portion of MB2848 was derived by using oligos
MO841 (SEQ ID NO:11) and MO2278 (SEQ ID NO:13) to PCR amplify the
lovE DNA binding domain from A. terreus cDNA. A second round of PCR
was performed with primers MO343 (SEQ ID NO:14) and MO2278 to add
appropriate Gateway Cloning Technology compatible sequences. The
At274 portion of MB2848 can be derived by using primers MO2273 (SEQ
ID NO:15) and MO2274 (SEQ ID NO:16) to PCR amplify the
carboxy-terminal domain of At274 from A. terreus cDNA. A second
round of PCR was performed with primers MO344 (SEQ ID NO:17) and
MO2273 to add appropriate Gateway Cloning Technology compatible
sequences. The lovE and At274 PCR products were cut with BamHI and
purified over a QIAquick PCR purification kit (Qiagen, Valencia,
Calif.) according to manufacturer's instructions. Finally, the
products were mixed 3-4 hours in a standard ligation reaction and
used in Gateway entry and destination reactions to create
MB2848.
[0115] Gateway cloning technology was used to clone the lovE
variants of interest into plasmid MB1419 which is a filamentous
fungal expression vector. The MB1419 fungal selection marker is the
A. nidulans GPD promoter controlling the ble gene from S.
hindustanus. The transgene is controlled by the A. nidulans PGK
promoter. A. terreus strain MF117 is a derivative of A. terreus
strain ATCC20542.
Example 2
[0116] PCR Mutagenesis of the lovE DNA Binding Domain
[0117] The zinc finger DNA binding domain of lovE is encoded by
nucleotides 100-201 (SEQ ID NO:92). Oligos MO2624 (SEQ ID NO:18)
and MO2654 (SEQ ID NO:19) were used to PCR amplify a lovE
containing fragment from plasmid MB2478. The 1.7 kb product
contains nucleotides 212-1410 of lovE and .about.500 bp of flanking
vector sequence. Two rounds of standard PCR (1.5 mM MgCl.sub.2)
were performed with Amplitaq DNA polymerase (Applied Biosystems,
Foster City, Calif.) according to the manufacturer's
instructions.
[0118] Plasmid MB2848 was cut with KpnI-BamHI to release a 1.1 kb
fragment containing the At274 portion of the lovE-At274 chimeric
open reading frame. The remaining 5.5 kb vector sequence retains
the lovE DNA binding domain.
Example 3
[0119] PCR Mutagenesis of the lovE Open Reading Frame
[0120] lovE open reading frame insert was prepared according to the
following procedure. Oligo pairs MO2680 (SEQ ID NO:20)/MO2686 (SEQ
ID NO:21), MO2681 (SEQ ID NO:22)/MO2686, and MO2700 (SEQ ID
NO:23)/MO2701 (SEQ ID NO:24) were used to PCR amplify the entire
lovE open reading frame from plasmid MB2478. The PCR products
differ in the amount of 5' and 3' vector sequence flanking the lovE
open reading frame.
[0121] PCR was performed using a GeneMorph PCR mutagenesis kit
(Stratagene, La Jolla, Calif.) according to manufacturer's
instructions to achieve medium and high range mutation
frequencies.
[0122] Plasmid MB2478 was cut with Asp718/XbaI to release a 1.7 kb
fragment. The remaining 5.0 kb vector sequence completely lacks
lovE ORF sequence.
Example 4
[0123] Transformation and Selection for G418R Isolates
[0124] All PCR products were purified using a QIAquick PCR
purification kit (Qiagen) according to manufacturer's instructions.
All vectors were gel purified using a QIAquick gel extraction kit
(Qiagen) according to manufacturer's instructions.
[0125] The mutagenesis strategy of Muhlrad et al. (Yeast 8:79-82
(1992)) was used which involves cotransforming a mutated PCR
product and gapped plasmids into S. cerevisiae, and then screening
for in vivo recombinants having the desired phenotype).
[0126] Transformation of Saccharomyces cerevisiae was accomplished
by the lithium acetate/single-stranded carrier DNA/polyethylene
glycol (LiAc/ss-DNA/PEG) protocol (Woods R. A. and Gietz R. D.
Methods Mol. Biol. 177:85-97 (2001)) with a 1:5 molar ratio of
vector:insert DNA to generate >55,000 in vivo recombinant
transformants on SC-Ura plates. Transformants were transferred by
replica printing to YPD plates containing 100 .mu.g/ml G418 and
allowed to grow for 2-4 days at 30.degree. C. (FIG. 1).
[0127] Drug resistant clones were confirmed in secondary assays
including growth on G418 concentrations up to 2000 .mu.g/ml. The
plasmid-dependence of the phenotype was determined by observing the
re-appearance of drug sensitivity correlating with loss of the
library plasmid. lovE variant plasmids were recovered from
promising candidates (Hoffman and Winston (1986) Gene 57:267). More
than 70 lovE variants were identified and definitively
characterized by DNA sequence and/or restriction digestion
analysis.
[0128] Table 3 summarizes the G418 resistance phenotype and
sequence analysis of 26 of these variants.
3TABLE 3 Variant lovE Mutations lovFp- MO oligos used Amino Amino
Amino Amino Amino Amino Amino Amino Amino Amino Amino neo for
random Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid lovE
Mediated PCR Change Change Change Change Change Change Change
Change Change Change Change allele G418R mutagenesis 1 2 3 4 5 6 7
8 9 10 11 1 -/+ 2624/2654 H253R S341P 2 +/- 2624/2654 R121W S133L
S322G 3 +++ 2624/2654 C73R A83V T135I 4 ++ 2624/2654 C73R E177G 5
++ 2624/2654 C73R 6 +/- 2624/2654 C153Y E197K T281A 7 + 2624/2654
C73R T256A N466S 8 +++ 2624/2654 C73R E141V 9 ++ 2624/2654 C73R
E303K 10 +++ 2624/2654 Q41K 16 +++ 2680/2686 Q14K P16A G23S T9M
Q362E 19 +/- 2700/2701 R21H S34A Q80H A84S E303D H374D A440T A441V
C445S P469S 20 + 2700/2701 F31L T409I 21 +++ 2700/2701 F31L M97I
E113D D146N P163S N367I H458Y 30 +/- 2681/2686 I43V Q295L 31 ++
2680/2686 F31L P101S C153R C159S E162K R293L S311N 32 ++ 2680/2686
L14I E18V G138C E338G V361L P389S N400S 33 ++ 2680/2686 Q41R S174Y
A402T 34 ++ 2680/2686 F31L T52I P101Q P108S V111I 36 +/- 2700/2701
D85N I143F M232I T315I S382Y M385K 37 ++ 2700/2701 T46I Q62R K77R
S323C N367Y V373I 38 -/+ 2700/2701 Q41R T294I P310L G337D P389L
A394V G436S 39 + 2680/2686 T52N V111I T139 V184I T281A 40 +++
2680/2686 Q41R D4E V87I D110E E141K A189T N276D T347R N367I Q377R
A425T 41 -/+ 2680/2686 D131N S133L R312G A429G wild- - N/A N/A
type
[0129] Table 4 summarizes amino acid substitutions that were
isolated multiple times, suggesting that they are particularly
important for improving lovE variant activity on lovFp-HIS3p-neo
expression.
4TABLE 4 lovE Mutations Isolated Multiple Times Number of Times
Amino Acid Change Isolated in lovE 1-41 lovE variant F31L 4 20, 21,
31, 34 Q41K 2* 10, 16 Q41R 3* 33, 38, 40 T52I/T52N 1 each 34, 39
C73R 6* 3, 4, 5, 7, 8, 9 P101S/P101Q 1 each 31, 34 V111I 2 34, 39
S133L 2 2, 41 E141V, E141K 1 each 8, 40 C153Y/C153R 1 each 6, 31
T281A 2 6, 39 N367I/N367Y 2/1 21, 40, 37 P389S/P389L 1 each 32, 38
*allele was isolated in additional lovE variants that were not
fully sequenced
Example 5
[0130] Increased lovF-lacZ Expression in S. cerevisiae
[0131] In order to quantify the increase in lovF expression,
.beta.-galactosidase activity was measured in lovE variant
transformed S. cerevisiae strains that also harbored lovFp-lacZ
reporter derivative plasmids. lovF-lacZ reporter derivative
plasmids were constructed as follows.
[0132] Plasmid MB1918 contains the lovFp-lacZ reporter gene. It can
be derived from pRS424 (Sikorski and Hieter (1989) Genetics
122:19-27). First, primers MO107 (SEQ ID NO:25) and MO197 (SEQ ID
NO:26) are used to PCR amplify the lacZ gene from Yep355 (Myers, et
al., Gene 45:299-310 (1986)). This lacZ-containing fragment was
inserted into the BamHI-HindIII sites of pRS416 (Sikorski and
Hieter, Genetics 122:19-27 (1989)). This same lacZ fragment can be
cut out of the resulting vector with KpnI-NotI and inserted into
the same sites of pRS424 to create pRS424-lacZ. Primers MO1293 (SEQ
ID NO:27) and MO1294 (SEQ ID NO:28) are used to PCR amplify a 2.09
kb fragment of the lovF promoter from A. terreus genomic DNA. The
lovF promoter fragment was then cut with NotI-BglII and inserted
into NotI-BamHI linearized pRS424-lacZ.
[0133] Plasmid MB2114 contains the lovFp-CYC1p-lacZ reporter gene.
It can be derived from pRS424-lacZ (see MB1918 plasmid
construction). Primers MO1787 (SEQ ID NO:29) and MO1788 (SEQ ID
NO:30) are used to amplify the 264 bp basal CYC1 element from
pRS415 CYC1 (Mumberg, et al., Gene 156:119-122 (1995)). This 264 bp
fragment was inserted upstream of the pRS424-lacZ derivative which
has been digested with SpeI-BamHI. Finally, the lovF promoter from
MB1918 was PCR amplified with MO1793 (SEQ ID NO:31) and MO1794 (SEQ
ID NO:32) and inserted into the NotI-SpeI sites to create
MB2114.
[0134] Yeast strains utilized in this study include strains MY2145
and MY2159, which are both derived from the S. cerevisiae sigma
1278b strain background; the genotypes are both strains are as
follows: MAT.alpha. ura3.DELTA.0 leu2.DELTA.0his3.DELTA.::hisG
trp1.DELTA.0::hisG. MY2145 and MY2159 contain the lovFp-lacZ
reporter plasmids MB2114 and MB1918, respectively.
[0135] MY2124 transformed with individual lovE variant plasmids was
mated to S. cerevisiae strains MY2154 and MY2159. Diploids were
selected on SC-UraTrp media. Multiple diploids from each individual
mating were assayed for lovFp-lacZ expression using 96 well format
.beta.-galactosidase assays. For .beta.-galactosidase assays, cells
were transferred from transformation plates to 96-well microtiter
plates containing 200 .mu.l Z buffer. 12 strains were transferred
simultaneously using a 12-channel multi-pipettor to scoop cells
from transformation plates. Duplicate samples were prepared for all
assays. OD.sub.600 readings were taken on samples in Z buffer.
These values were used to normalize for equal cell number in all
assays. After determining OD.sub.600, 150 .mu.l of each sample in Z
buffer was transferred onto a Millipore Multiscreen Assay System
(Nitrocellulose Immobilon NC), filtered, and then washed by
filtering 200 .mu.l Z buffer. 100 .mu.l Z buffer with .beta.ME and
detergents was then added to each well, as was 20 .mu.l 4 mg/ml
ONPG. Reactions were incubated at 30.degree. C., stopped with 50
.mu.l 1 M Na.sub.2CO.sub.3, filtered into a polystyrene 96-well
assay plate, and OD.sub.420 was determined for each assay well.
.beta.-galactosidase units were determined using the Miller formula
(O.D. 420.times.1000)/(OD600*minutes*volume in mL). Z buffer is
made by dissolving the following in 1 L of water (16.1 g
Na.sub.2HPO.sub.4-7H.sub- .2O, 5.5 g NaH.sub.2PO.sub.4-H.sub.2O,
0.75 g KCl and 0.246 g MgSO.sub.4-7H.sub.2O). Z buffer with
detergents and .beta.ME is made as follows: 9.8 ml Z buffer, 100
.mu.l 20 mg/ml CTAB, 100 .mu.l 10 mg/ml sodium deoxycholate, and 69
.mu.l .beta.ME Control plasmids utilized in these studies included
MB968, MB2478 and MB1644.
[0136] Results of these studies are presented in FIGS. 2-5,
demonstrating increased transcription-activating properties of the
lovE variants disclosed herein.
Example 6
[0137] Secondary Metabolite Production
[0138] Transformation of filamentous fungi was performed according
to the following procedure. Protoplasts were generated by
inoculating rich media with spores. Spores were allowed to
germinate for about 20 hrs or until germ tubes were between 5 and
10 spore lengths. The germlings were centrifuged and washed twice
with sterile distilled water and once with 1 M magnesium sulfate.
Germlings were then resuspended in 1M magnesium sulfate containing
approximately 2 mg/ml of Novozyme. Tubes were then incubated at
30.degree. C. shaking at 80 RPM for about 2 hrs or until most of
the hyphae were digested and protoplasts were abundant. Protoplasts
were filtered through one layer of Miracloth. At least one volume
of STC was added and protoplasts were centrifuged. Protoplasts were
washed twice with STC. Protoplasts then were resuspended in 1 ml
STC and counted in a hemacytometer. A final concentration of
approximately 5.times.10.sup.7 protoplasts/ml were frozen in a
9:1:0.1 solution of STC, SPTC and DMSO in a Nalgene Cryo cooler at
-80.degree. C. (cools -1.degree. C./min).
[0139] Solutions for transformation were as follows: STC (0.8 M
Sorbitol, 25 mM Tris-HCl pH 7.5, 25 mM CaCl.sub.2) and SPTC (0.8 M
Sorbitol, 40% PEG 4000, 25 mM Tris-HCl pH 8, 50 mM CaCl.sub.2).
Transformation was accomplished according to the following
protocol. 1-5 .mu.g of DNA comprising a lovE variant according to
the invention in a fungal expression vector was placed in a 50 ml
Falcon tube. 100 .mu.l of previously frozen protoplasts were added
to the DNA, gently mixed, and then incubated on ice for 30 min. 15
.mu.l of SPTC was added, followed by mixing by tapping and
incubation at RT for 15 min. 500 .mu.l SPTC was added and mixed
well by tapping and rolling, then incubated at RT for 15 min. 25
mls of regeneration minimal medium was added, mixed well and poured
on plates containing 25 mls of regeneration minimal medium with
2.times. the concentration of selection drug.
[0140] Transformation plates were incubated at 26.degree. C. for
5-6 days or until colonies started to appear. Regeneration minimal
medium contains trace elements, salts, 25 mM sodium nitrate, 0.8 M
Sucrose, and 1% agarose at pH 6.5. The selection drug that was used
successfully with A. terreus is phleomycin, a broad-spectrum
glycopeptide antibiotic. Transformants were picked onto new plates
with a toothpick (if the fungus was sporulating) or with sterile
forceps (if the fungus did not sporulate). Purification plates
contained minimal medium (same as regeneration minimal medium but
containing 2% instead of 0.8 M sucrose) and 1.times. drug
concentration. Picked transformants were incubated at 26.degree. C.
for 5-6 days.
[0141] Transformants were grown in production media for secondary
metabolite production. Briefly, for A. terreus and lovastatin
production, spores were used as the inoculum. Spores were obtained
from the purification plate by using a wooden inoculation stick.
The medium was RPM containing corn steep liquor, sodium nitrate,
potassium phosphate, magnesium sulfate, sodium chloride, P2000 (Dow
chemical), trace elements and lactose or glucose as carbon source.
The medium was pH 6.5. Flasks were incubated at 26.degree. C. with
shaking at 225 RPM. For static 96-well cultures, the same medium
was used and the spores were obtained from the purification plate
with a wooden toothpick. 96-well plates were incubated, without
shaking at 26.degree. C.
[0142] Sampling was done after after 5 days for lovastatin.For
shake flask experiments 1-1.5 mls of supernatant was placed into
96-well plates, which were centrifuged and supernatants transferred
to new 96-well plates. Samples were frozen at .sup.-80.degree. C.
for storage or for later assays.
[0143] Cultures that were grown standing in a 96-well plate were
centrifuged and the supernatant was transferred to a new 96 well
plate. Samples were frozen at .sup.-80.degree. C.
Example 7
[0144] Measurement of Secondary Metabolite Production
[0145] The concentration of the secondary metabolite lovastatin was
determined by enzyme inhibition assay (FIG. 6). Briefly, 10 .mu.L
of sample was removed and diluted 1:100 in H.sub.2O. 10 .mu.l of
this diluted broth was assayed in a reaction (200 .mu.L total)
containing 1 mM HMGCOA, 1 mM NADPH, 0.005 mM DTT and 5 .mu.l (His)
.sub.6HMGR. The disappearance of absorbance at 340 nm was observed
over time. This represents the disappearance of NADPH, and
lovastatin inhibits this reaction.
[0146] The initial velocities were calculated for the reactions
containing samples, adjusted for dilution, and compared to
reactions containing lovastatin standards to determine levels of
metabolite produced. (His) .sub.6HMGR was expressed in
Saccharomyces cerevisiae and purified with a nickel column.
[0147] The results from ten individual transformants for each
allele are shown in standard box plot format in FIG. 6. Lovastatin
concentration from the corresponding wild-type lovE control is
shown in matching fill pattern. For example, lovE alleles 2, 7, 8
and 9 were all transformed and assayed at the same time as the
non-hatched wild-type control. The horizontal line in each
individual box represents the median.
[0148] Lovastatin concentration was also determined by high
pressure liquid chromatography (HPLC). Briefly, 100 .mu.L of broth
sample was removed and diluted 1:10 into 70% H.sub.2O-30%
acetonitrile (900 .mu.l). This mixture was spun down to pellet
debris at 13000 RPM for 5 minutes. 900 .mu.l of this diluted broth
was transferred to a vial and the sample was analyzed by HPLC. 10
.mu.l were injected into a Waters HPLC system (996 photo-diode
array detector, 600 E pump controller and 717 autosampler) equipped
with a YMC-Pack ODS column (Aq-302-3, 150.times.4.6 mm ID, S-3
.mu.M pore size) and eluted with isocratic 40% aqueous acetic acid
(0.7%)-60% acetonitrile for 8 minutes. Lovastatin was detected at
238 nm to have a retention time of 6.5 minutes and was quantified
using a calibration curve created from pure lovastatin samples.
[0149] The results from ten individual transformants for each lovE
variant are shown in standard box plot format in FIGS. 7A and 7B.
Thirty individual wild-type lovE transformants and ten individual
MB2143 negative control transformants were tested. Identical
controls are plotted in FIGS. 7A and 7B.
[0150] PCR analysis of A. terreus transformants demonstrates that
greater than fifty percent of the transformants contain the
transgene. Variability in levels of transgene expression can
presumably be influenced by integration site and copy number. lovE
variants containing identical amino acid substitutions are
labeled.
[0151] The amino acid and nucleic acid sequences of lovE variant
sequences are presented in Table 5 and Table 6, respectively.
5Table 5 Amino Acid Sequences of Variants of the lovE Gene lovE-1
maadggiftnsvtlspvegsrtggt- lprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:41)
rcvysercpkrklrqsraadlvsadpdpclhmssppvpsqslpldvseshssntsrqfldppdsy
dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspfestvekaplppvssdiaraasaq
relfddlsavsqeleeillavtvewpkqeiwthpigmffnasrrlltvlrqqaqadc- rqgtldec
lrtknlftavhcyilnvriltaiselllsqirrtqnshmsplegsrsqs- psrddtssssghssvd
tipffsenlpigelfpyvdplthalfsacttlhvgvqllre- neitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsde- gafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-2
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgr- apcqrcqqagl
(SEQ ID NO:42) rcvysercpkrklrqsraadlvsadpdpclh-
mssppvpsqslpldvseshssntswqfldppdsy dwlwtsigtdeaidtdcwglsqc-
dggfscqleptlpdlpspfestvekaplppvssdiaraasaq
relfddlsavsqeleeillavtvewpkqeiwthpigmffnasrrlltvlrqqaqadchqgtldec
lrtknlftavhcyilnvriltaiselllsqirrtqnshmsplegsrsqspsrddtssssghgsvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaas- ismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrti- aalrrcyedifslark
hkhgmlrdlnnipp lovE-3
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:43) rcvyserrpkrklrqsrvadlvsadpdpclhmssppvpsqslpldvs-
eshssntsrqfldppdsy dwswisigtdeaidtdcwglsqcdggfscqleptlpdlp-
spfestvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiw-
thpigmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltai-
selllsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-4
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:44) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvgkaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-5
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:45) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-6
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:46) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-7
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:47) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-8
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:48) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-9
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:49) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-10
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:50) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-16
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:51) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-19
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:52) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-20
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:53) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-21
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:54) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-30
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:55) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-31
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:56) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-32
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:57) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-33
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:58) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-34
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:59) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-36
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:60) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-37
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:61) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-38
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:62) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-39
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:63) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-40
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:64) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp lovE-41
maadqgiftnsvtlspvegsrtggtlprrafrrscdrchaqkikctgnkevtgrapcqrcqqagl
(SEQ ID NO:65) rcvyserrpkrklrqsraadlvsadpdpclhmssppvpsqslpldvsesh-
ssntsrqfldppdsy dwswtsigtdeaidtdcwglsqcdggfscqleptlpdlpspf-
estvekaplppvssdiaraasaq relfddlsavsqeleeillavtvewpkqeiwthp-
igmffnasrrlltvlrqqaqadchqgtldec lrtknlftavhcyilnvriltaisel-
llsqirrtqnshmsplegsrsqspsrddtssssghssvd
tipffsenlpigelfsyvdplthalfsacttlhvgvqllreneitlgvhsaqgiaasismsgepg
ediartgatnsarceeqpttpaarvlfmflsdegafqeaksagsrgrtiaalrrcyedifslark
hkhgmlrdlnnipp
[0152]
6Table 6 DNA Sequences of Variants of the lovE Gene lovE-1
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGT- CACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:66)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-2
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:67)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-3
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:68)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-4
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:69)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-5
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:70)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-6
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:71)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-7
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:72)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-8
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:73)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-9
ATGGCTGCAGATCAAGGTATATT- CACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:74)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-10
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:75) CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGT-
CATGCACAAAAGATCA AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGT-
CAGCGTTGCCAGCAGGCTGGACTT CGATGCGTCTACAGTGAGCGATGCCCCAAGCGC-
AAGCTACGCCAATCCAGGGCAGCGGATCTCGT CTCTGCTGACCCAGATCCCTGCTTG-
CACATGTCCTCGCCTCCAGTGCCCTCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCCAACGCTGCCGGATCTACCTTCGCC- CTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCG- TGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACT- GGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCA- TCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACA- AGCGCAGGCCGACTGCCGTCAAGGCACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACACCAGCAGCAGCAGCGGCCACAGCA- GTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCT- ATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGG- TACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTG- CAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGA- CCAATTCCGCAAGATGCGAGGAGCAGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAATATTCCTCCATGA lovE-16
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAG- GGTTCACGCAC
(SEQ ID NO:76) CGGTGGAACATTACCCCGCCGTGCATTCCG-
ACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCGGATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCCTCACAGAG- CTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGA- TCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGA- CACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCC- AACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCC- ACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGGCACACTAG- ACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATG- TGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGA- ACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACA- CCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACC- TCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGCAGCCGACC- ACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAG- GCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAG- GATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAAT- ATTCCTCCATGA lovE-19
ATGGCTGCAGATCAAGGTATATTCACGAAC- TCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:77)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-20
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:78) CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGT-
CATGCACAAAAGATCA AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGT-
CAGCGTTGCCAGCAGGCTGGACTT CGATGCGTCTACAGTGAGCGATGCCCCAAGCGC-
AAGCTACGCCAATCCAGGGCAGCGGATCTCGT CTCTGCTGACCCAGATCCCTGCTTG-
CACATGTCCTCGCCTCCAGTGCCCTCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCCAACGCTGCCGGATCTACCTTCGCC- CTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCG- TGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACT- GGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCA- TCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACA- AGCGCAGGCCGACTGCCGTCAAGGCACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACACCAGCAGCAGCAGCGGCCACAGCA- GTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCT- ATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGG- TACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTG- CAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGA- CCAATTCCGCAAGATGCGAGGAGCAGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAATATTCCTCCATGA lovE-21
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAG- GGTTCACGCAC
(SEQ ID NO:79) CGGTGGAACATTACCCCGCCGTGCATTCCG-
ACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCGGATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCCTCACAGAG- CTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGA- TCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGA- CACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCC- AACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCC- ACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGGCACACTAG- ACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATG- TGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGA- ACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACA- CCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACC- TCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGCAGCCGACC- ACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAG- GCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAG- GATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAAT- ATTCCTCCATGA lovE-30
ATGGCTGCAGATCAAGGTATATTCACGAAC- TCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:80)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-31
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:81) CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGT-
CATGCACAAAAGATCA AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGT-
CAGCGTTGCCAGCAGGCTGGACTT CGATGCGTCTACAGTGAGCGATGCCCCAAGCGC-
AAGCTACGCCAATCCAGGGCAGCGGATCTCGT CTCTGCTGACCCAGATCCCTGCTTG-
CACATGTCCTCGCCTCCAGTGCCCTCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCCAACGCTGCCGGATCTACCTTCGCC- CTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCG- TGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACT- GGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCA- TCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACA- AGCGCAGGCCGACTGCCGTCAAGGCACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACACCAGCAGCAGCAGCGGCCACAGCA- GTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCT- ATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGG- TACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTG- CAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGA- CCAATTCCGCAAGATGCGAGGAGCAGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAATATTCCTCCATGA lovE-32
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAG- GGTTCACGCAC
(SEQ ID NO:82) CGGTGGAACATTACCCCGCCGTGCATTCCG-
ACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCGGATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCCTCACAGAG- CTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGA- TCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGA- CACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCC- AACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCC- ACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGGCACACTAG- ACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATG- TGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGA- ACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACA- CCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACC- TCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGCAGCCGACC- ACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAG- GCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAG- GATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAAT- ATTCCTCCATGA lovE-33
ATGGCTGCAGATCAAGGTATATTCACGAAC- TCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:83)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-34
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:84) CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGT-
CATGCACAAAAGATCA AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGT-
CAGCGTTGCCAGCAGGCTGGACTT CGATGCGTCTACAGTGAGCGATGCCCCAAGCGC-
AAGCTACGCCAATCCAGGGCAGCGGATCTCGT CTCTGCTGACCCAGATCCCTGCTTG-
CACATGTCCTCGCCTCCAGTGCCCTCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCCAACGCTGCCGGATCTACCTTCGCC- CTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCG- TGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACT- GGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCA- TCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACA- AGCGCAGGCCGACTGCCGTCAAGGCACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACACCAGCAGCAGCAGCGGCCACAGCA- GTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCT- ATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGG- TACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTG- CAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGA- CCAATTCCGCAAGATGCGAGGAGCAGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAATATTCCTCCATGA lovE-36
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAG- GGTTCACGCAC
(SEQ ID NO:85) CGGTGGAACATTACCCCGCCGTGCATTCCG-
ACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCGGATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCCTCACAGAG- CTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGA- TCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGA- CACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCC- AACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCC- ACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGGCACACTAG- ACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATG- TGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGA- ACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACA- CCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACC- TCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGCAGCCGACC-
ACTCCAGC GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAG-
GCAAAGTCTGCTGGTT CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAG-
GATATCTTTTCCCTCGCCCGCAAA CACAAACATGGCATGCTCAGAGACCTCAACAAT-
ATTCCTCCATGA lovE-37 ATGGCTGCAGATCAAGGTATATTCACGAAC-
TCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC (SEQ ID NO:86)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-38
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:87) CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGT-
CATGCACAAAAGATCA AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGT-
CAGCGTTGCCAGCAGGCTGGACTT CGATGCGTCTACAGTGAGCGATGCCCCAAGCGC-
AAGCTACGCCAATCCAGGGCAGCGGATCTCGT CTCTGCTGACCCAGATCCCTGCTTG-
CACATGTCCTCGCCTCCAGTGCCCTCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCCAACGCTGCCGGATCTACCTTCGCC- CTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCG- TGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACT- GGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCA- TCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACA- AGCGCAGGCCGACTGCCGTCAAGGCACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACACCAGCAGCAGCAGCGGCCACAGCA- GTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCT- ATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGG- TACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTG- CAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGA- CCAATTCCGCAAGATGCGAGGAGCAGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAATATTCCTCCATGA lovE-39
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAG- GGTTCACGCAC
(SEQ ID NO:88) CGGTGGAACATTACCCCGCCGTGCATTCCG-
ACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCGGATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCCTCACAGAG- CTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGA- TCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGA- CACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCC- AACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCC- ACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGGCACACTAG- ACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATG- TGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGA- ACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACA- CCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACC- TCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGCAGCCGACC- ACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAG- GCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAG- GATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAAT- ATTCCTCCATGA lovE-40
ATGGCTGCAGATCAAGGTATATTCACGAAC- TCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:89)
CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGTCATGCACAAAAGATCA
AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGTCAGCGTTGCCAGCAGGCTGGACTT
CGATGCGTCTACAGTGAGCGATGCCCCAAGCGCAAGCTACGCCAATCCAGGGCAGCG- GATCTCGT
CTCTGCTGACCCAGATCCCTGCTTGCACATGTCCTCGCCTCCAGTGCCC- TCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAG- TTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAG- GCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAG- TTACAGCCAACGCTGCCGGATCTACCTTCGCCCTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCGTGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACTGGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCATCCCATCGGAATGTTTTTCAATGC- GTCACGAC
GGCTTCTTACTGTCCTGCGCCAACAAGCGCAGGCCGACTGCCGTCAAGG- CACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACAT- ATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCG- GACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAG- AGACGACACCAGCAGCAGCAGCGGCCACAGCAGTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCTATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGGTACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTGCAGCTTCCATCAGCATGAGCGGGG- AACCAGGC
GAGGATATAGCCAGGACAGGGGCGACCAATTCCGCAAGATGCGAGGAGC- AGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTT- TCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGAT- GCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACC- TCAACAATATTCCTCCATGA lovE-41
ATGGCTGCAGATCAAGGTATATTCACGAACTCGGTCACTCTCTCGCCAGTGGAGGGTTCACGCAC
(SEQ ID NO:90) CGGTGGAACATTACCCCGCCGTGCATTCCGACGCTCTTGTGATCGGTGT-
CATGCACAAAAGATCA AATGTACTGGAAATAAGGAGGTTACTGGCCGTGCTCCCTGT-
CAGCGTTGCCAGCAGGCTGGACTT CGATGCGTCTACAGTGAGCGATGCCCCAAGCGC-
AAGCTACGCCAATCCAGGGCAGCGGATCTCGT CTCTGCTGACCCAGATCCCTGCTTG-
CACATGTCCTCGCCTCCAGTGCCCTCACAGAGCTTGCCGC
TAGACGTATCCGAGTCGCATTCCTCAAATACCTCCCGGCAGTTTCTTGATCCACCGGACAGCTAC
GACTGGTCGTGGACCTCGATTGGCACTGACGAGGCTATTGACACTGACTGCTGGGGGCTGTCCCA
ATGTGATGGAGGCTTCAGCTGTCAGTTACAGCCAACGCTGCCGGATCTACCTTCGCC- CTTCGAGT
CTACGGTTGAAAAAGCTCCGTTGCCACCGGTATCGAGCGACATTGCTCG- TGCGGCCAGTGCGCAA
CGAGAGCTTTTCGATGACCTGTCGGCGGTGTCGCAGGAACT- GGAAGAGATCCTTCTGGCCGTGAC
GGTAGAATGGCCGAAGCAGGAAATCTGGACCCA- TCCCATCGGAATGTTTTTCAATGCGTCACGAC
GGCTTCTTACTGTCCTGCGCCAACA- AGCGCAGGCCGACTGCCGTCAAGGCACACTAGACGAATGT
TTACGGACCAAGAACCTCTTTACGGCAGTACACTGTTACATATTGAATGTGCGGATTTTGACCGC
CATATCGGAGTTGCTCCTGTCGCAAATTAGGCGGACCCAGAACAGCCATATGAGCCCACTGGAAG
GGAGTCGATCCCAGTCGCCGAGCAGAGACGACACCAGCAGCAGCAGCGGCCACAGCA- GTGTTGAC
ACCATACCCTTCTTTAGCGAGAACCTCCCTATTGGTGAGCTGTTCCCCT- ATGTTGACCCCCTGAC
ACACGCCCTATTCTCGGCTTGCACTACGTTACATGTTGGGG- TACAATTGCTGCGTGAGAATGAGA
TTACTCTGGGAGTACACTCCGCCCAGGGCATTG- CAGCTTCCATCAGCATGAGCGGGGAACCAGGC
GAGGATATAGCCAGGACAGGGGCGA- CCAATTCCGCAAGATGCGAGGAGCAGCCGACCACTCCAGC
GGCTCGGGTTTTGTTCATGTTCTTGAGTGATGAAGGGGCTTTCCAGGAGGCAAAGTCTGCTGGTT
CCCGAGGTCGAACCATCGCAGCACTGCGACGATGCTATGAGGATATCTTTTCCCTCGCCCGCAAA
CACAAACATGGCATGCTCAGAGACCTCAACAATATTCCTCCATGA
Equivalents
[0153] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompasssed by the
following claims.
* * * * *